Compositions and methods for treating and preventing inflammation

ABSTRACT

The present invention provides novel compounds compositions and methods for (i) treating or preventing inflammation; and (ii) preventing or reducing hyperactivation of innate immune response, by inhibiting NRP1-dependent cell-signaling. Also provided are compounds, composition, and methods of specifically inhibiting SEMA3A-mediated cell signaling.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application No. 16/422,273, filed May 24,2019, which is a divisional of U.S. application Ser. No. 15/507,407,filed Feb. 28, 2017, issued as U.S. Pat. No. 10,738,122 on Aug. 11,2020, which is the U.S. National Stage of International PatentApplication No. PCT/CA2015/050862, filed Sep. 8, 2015, which waspublished in English under PCT Article 21(2), which in turn claimspriority of U.S. provisional application Ser. No. 62/046,459, filed onSep. 5, 2014. The above-referenced applications are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formentitled “98565-03_ST25.txt”, created on Jul. 30, 2020 having a size of579 KB. The computer readable form is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N.A.

FIELD OF THE INVENTION

The present invention relates to inflammation. More specifically, thepresent invention is concerned with the inhibition of the NRP1 pathwayfor the prevention or treatment of inflammation.

REFERENCE TO SEQUENCE LISTING

N.A.

BACKGROUND OF THE INVENTION

Local acute inflammatory responses are predominantly beneficial andconstitute the body's first line of defense against infection of thehost. Conversely, acute systemic inflammation such as in septic shock isa leading cause of morbidity and mortality (58). When chronic, low-gradeinflammation persists, it can be at the origin of a several systemicdiseases ranging from type II Diabetes Mellitus, arthritis, cancer, anumber of neuro-inflammatory conditions and more.

Of all cytokines, receptors and other players thought to contribute tothe inflammatory processes, one paradigm that has been largelyoverlooked is the influence of classical neuronal guidance cues andtheir receptors. These include semaphorin3A (SEMA3A, e.g., mRNA:NM_006080; and protein: NP_006071 and FIG. 21) and their receptorNeuropilin-1 (NRP1, e.g., mRNA: NM_001024628; and protein: NP_001019799,NM_003873 and FIGS. 22 (isoform 2 or b, secreted) and 26 (isoform 1).NRP1 is expressed on both lymphoid and myeloid cells (59, 31). Yet itsrole in inflammation is largely unknown and especially in the context ofcytokine production.

The Semaphorins were initially characterized as key players in axonalguidance during embryogenesis. It is now clear that the role ofSemaphorins extends beyond axonal guidance and influence vascularsystems, tumor growth and the immune response. The Semaphorin familycounts at least 21 vertebrate genes and 8 additional genes ininvertebrates. All Semaphorins contain a ˜500 amino acid SEMA domainthat is required for signaling. Class 3 Semaphorins (such as SEMA3A) arethe only secreted members of the family. SEMA3A is synthesized as adisulphide-linked homodimer and dimerization is essential for signaling.

In neurons, binding of SEMA3A to its cognate receptor Neuropilin-1(NRP1) provokes cytoskeletal collapse via plexins (60); the transductionmechanism in endothelial cells remains ill-defined. NRP1 has theparticular ability to bind two structurally dissimilar ligands viadistinct sites on its extracellular domain (27-29). It binds not onlySEMA3A (46, 47) provoking cytoskeletal collapse but also VEGF₁₆₅ (28,29, 47, 61) enhancing binding to VEGFR2 and thus increasing itsangiogenic potential (62). Crystallographic evidence revealed thatVEGF₁₆₅ and SEMA3A do not directly compete for NRP1 but rather cansimultaneously bind to NRP1 at distinct, non-overlapping sites (63).Moreover, genetic studies show that NRP1 distinctly regulates theeffects of VEGF and SEMA3A on neuronal and vascular development (64).Finally, NRP1 has also been found to bind to TGF-β1 and to regulate itslatent form.

NRP1 is a single-pass transmembrane receptor with a large 860 amino acidextracellular domain subdivided into 3 sub-domains (a1a2, b1b2 and c)and a short 40 amino acid intracellular domain (65). In neurons, bindingof SEMA3A to NRP1 recruits Plexins, which transduce their intracellularsignal (60) and provoke cytoskeletal collapse. The transductionmechanism in endothelial cells remains ill-defined. NRP1 binds SEMA3A(46, 47) primarily via its a1a2 (but possibly also b1-) domain(provoking cytoskeletal collapse) and VEGF₁₆₅(28, 29, 47, 61)via itsb1b2 domain (enhancing binding to VEGFR2 and thus increasing itsangiogenic potential (62). The elevated levels of SEMA3A in the ischemicretina may thus partake in forcing neovessels into the vitreous bycollapsing and deviating the advancing tip cells away from the source ofthe repellent cue (21).

The CNS had long been considered an immune-privileged system, yet it isnow clear that the brain, retina and spinal cord are subjected tocomplex immune-surveillance (1, 2). Immunological activity in the CNS islargely dependent on an innate immune response and is present in healthand heightened in diseases such as diabetic retinopathy, multiplesclerosis, amyotrophic lateral sclerosis and Alzheimer's disease. Thisis apparent in the retina where an intensified, largelymicroglial/macrophage-based immune response is associated with theprogression of several sight threatening diseases such as diabeticretinopathy (DR)(3-5), age related macular degeneration (AMD)(6-8) andretinopathy of prematurity (ROP)(9, 10). Together, these retinaldiseases account for the principal causes of loss of sight inindustrialized countries (6, 11, 12).

Many of the current line of treatments of inflammatory diseases andconditions suffer from important side-effects and deficient long-termsafety profiles. Accordingly, there remains a need for novelpharmaceutical targets and methods of treatments.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present inventors have sought to determine the function ofmyeloid-resident NRP1 in the context of the innate immune response.

The present inventors have determined that SEMA3A, VEGF and TGF-β act aspotent attractants for mononuclear phagocytes (MPs, e.g., microglia andmacrophages) expressing the NRP1 receptor. Inhibition of NRP1 signalingin innate immune cells was shown to result in protection againstMPs-dependent inflammation and tissue damage under a variety ofconditions involving hyperactivation of the innate immune-responseincluding proliferative retinopathies, septic shock and cerebralischemia/stroke. Furthermore, the present inventors have designedvarious soluble NRP1-derived traps which inhibit SEMA3A signalling andshown that inhibition of SEMA3A significantly reduce the inflammatoryresponse in various conditions.

Accordingly, the present invention relates to the inhibition of NRP1cell signalling (e.g., NRP1 and its ligands) for the prevention ortreatment of inflammatory diseases and conditions involvinghyperactivation (i.e., pathological activation) of the innate immuneresponse. Non-limiting examples of such disease and conditions includesepsis, stroke, cerebral ischemia, and various proliferativeretinopathies.

More specifically, in an aspect, the present invention concerns a methodof treating or preventing inflammation comprising inhibitingNRP1-dependent cell-signaling.

In another aspect, the present invention relates to a method ofpreventing or reducing hyperactivation of innate immune responsecomprising inhibiting NRP1-dependent cell-signaling. In an embodiment,the hyperactivation of innate immune response comprises i) secretion ofIL-1β and TNFα and/or activation/recruitment of mononuclear phagocytes(MPs).

In an embodiment, inhibiting NRP1-dependent cell-signaling comprises: a)reducing NRP1 expression or activity; and/or b) reducing NRP1 ligandexpression or activity. In an embodiment, the NRP1 ligand is SEMA3A,VEGF₁₆₅ or TGF-β. In a particular embodiment, the NRP1 ligand is SEMA3A.

In an embodiment, reducing NRP1 activity consists of inhibiting thebinding of NRP1 to at least one NRP1 ligand. In an embodiment,inhibiting the binding of NRP1 to at least one NRP1 ligand comprisesadministering an NRP1 antibody (e.g., a SEMA3A antibody).

In another embodiment of the above methods, reducing NRP1 activitycomprises administering an effective amount of an NRP1 trap whichcomprises soluble NRP1 polypeptide or a functional fragment thereof. Ina particular embodiment, the NRP1 trap is as set forth in FIG. 19 or 20.

In a particular embodiment, the NRP1 trap of the present inventioninhibits the binding of SEMA3A to NPR-1 but does not substantiallyinhibit the binding of VEGF to NRP1. In an embodiment, such NRP1 trapcomprises the a1a2 domain of NRP1 but does not comprise the b1 and/or b2subdomain(s) of NRP1. In another embodiment, such trap comprises amutation in domain b1 at a position corresponding to tyrosine 297 of theNRP1 amino acid sequence as set forth in FIG. 22 which reduces orabrogates VEGF binding to the trap. In a specific embodiment, themutation changes the tyrosine at position 297 to an alanine.

In specific embodiments, the NRP1 trap of the present invention: a)comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domainsa1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1;d) comprises domains al and a2 of NRP1; e) comprises domain b1, whereinthe b1 domain comprises a mutation in amino acid corresponding totyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f)comprises domain b1, wherein the b1 domain comprises a mutation in aminoacid corresponding to tyrosine 297 of NRP1 which changes the tyrosine toan alanine; g) does not comprise domain c of NRP1; h) does not comprisedomain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j)does not comprise domains b1, b2 and c of NRP1.

In an embodiment of the above methods, inhibiting NRP1 ligand expressionor activity comprises specifically inhibiting SEMA3A expression orSEMA3A binding to NRP1. In a particular embodiment, inhibiting SEMA3Abinding to NRP1 comprises administering a SEMA3A antibody.

In a particular embodiment, the method of the present inventioncomprises reducing NRP1 expression by administering a NRP1 antisense,shRNA or siRNA.

In another embodiment, the method comprises reducing SEMA3A expressionby administering a SEMA3A antisense, shRNA or siRNA.

In a further aspect, the present invention concerns a compound for theprevention or treatment of inflammation wherein the compound a) reducesNRP1 expression or activity; or b) reduces NRP1 ligand expression oractivity.

In another aspect, the present invention relates to a compound forpreventing or reducing hyperactivation of innate immune response,wherein the compound a) reduces NRP1 expression or activity; or b)reduces NRP1 ligand expression or activity.

In an embodiment, the compound is: i) A SEMA3A antibody; ii) A NRP1antibody; iii) A NRP1 trap; iv) A SEMA3A antisense, shRNA or siRNA; orv) A NRP1 antisense, shRNA or siRNA. In another particular embodimentthe compound is a NRP1 antibody or a NRP1 trap and said compound doesnot substantially reduce the binding of VEGF to NRP1.

In a particular embodiment, the compound is a NRP1 trap. In anembodiment, the NRP1 trap is as set forth in FIGS. 19, 20, 27 and Table1.

In another embodiment, the NRP1 trap of the present invention inhibitsthe binding of SEMA3A to NPR-1 but does not substantially inhibit thebinding of VEGF to NRP1. In an embodiment, such NRP1 trap comprises thea1a2 domain of NRP1 but does not comprise the b1 and/or b2 subdomain(s)of NRP1. In another embodiment, such trap comprises a mutation in domainb1 at a position corresponding to tyrosine 297 of the NRP1 amino acidsequence as set forth in FIG. 22 which reduces or abrogates VEGF bindingto the trap. In a specific embodiment, the mutation changes the tyrosineat position 297 to an alanine.

In specific embodiments, the NRP1 trap of the present invention: a)comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domainsa1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1;d) comprises domains al and a2 of NRP1; e) comprises domain b1, whereinthe b1 domain comprises a mutation in amino acid corresponding totyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f)comprises domain b1, wherein the b1 domain comprises a mutation in aminoacid corresponding to tyrosine 297 of NRP1 which changes the tyrosine toan alanine; g) does not comprise domain c of NRP1; h) does not comprisedomain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j)does not comprise domains b1, b2 and c of NRP1.

In an embodiment, the NRP1 trap of the present invention comprises: (i)amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide setforth in FIG. 26 (SEQ ID NO: 69); (ii) amino acids 1 to 583 (preferably22 to 583) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69);(iii) amino acids 1 to 424 (preferably 22-424) the NRP1 polypeptide setforth in FIG. 26 (SEQ ID NO: 69); (iv) amino acids 1 to 265 (preferably22 to 265) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69);(v) 1 to 430 and 584 to 856 (preferably 22-430 and 584-856) the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vi) amino acids 1 to274 and 584 to 856 (preferably 22-274 and 584 to 856) the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vii) amino acids 1 to430 and 584 (preferably 22 to 430 and 584 to 856) of the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69). In a particularembodiment, the above noted traps comprise one or more mutation toreduce VEGF or SEMA3A binding as described above.

In another aspect, the present invention provides compositions for i)treating and preventing inflammation or ii) for preventing or reducingthe hyperactivation of the innate immune response, comprising one ormore compounds of the present invention together with a pharmaceuticalcarrier.

The present invention also relates to the use of one or more compoundsof the present invention in the manufacture of a medicament for i)treating and preventing inflammation or ii) for preventing or reducingthe hyperactivation of the innate immune response.

In a related aspect, the present invention concerns the use of one ormore compounds of the present invention for i) treating and preventinginflammation or ii) for preventing or reducing the hyperactivation ofthe innate immune response.

In a particular embodiment, the methods, compounds (e.g., NRP1polypeptide traps, nucleic acids encoding same, vectors, cellscomprising vectors, etc.), compositions and uses of the presentinvention are for treating or preventing inflammatory diseases andconditions selected from the group consisting of septic shock,arthritis, inflammatory bowel disease (IBD), cutaneous skininflammation, diabetes, uveitis, diabetic retinopathy, age-relatedmacular degeneration (AMD), retinopathy of prematurity, multiplesclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitivedecline/Alzheimer's disease or stroke.

In an embodiment, the methods, compounds, compositions and uses of thepresent invention are for treating or preventing septic shock, cerebralischemia or stroke.

More specifically, in accordance with the present invention, there isprovided the following items:

1. A method of treating or preventing inflammation comprising inhibitingNRP1-dependent cell-signaling in a subject.

2. A method of preventing or reducing hyperactivation of innate immuneresponse comprising inhibiting NRP1-dependent cell-signaling in asubject.

3. The method of item 2, wherein said hyperactivation of innate immuneresponse comprises i) secretion of IL-6, IL-1β and TNFα and/orrecruitment of mononuclear phagocytes (MPs).

4. The method of any one of items 1-3, wherein inhibiting NRP1-dependentcell-signaling comprises: (a) reducing NRP1 expression or activity;and/or (b) reducing NRP1 ligand expression or activity; wherein saidNRP1 ligand is SEMA3A, VEGF and/or TGF-β.

5. The method of item 4, wherein the method comprises (i) reducing NRP1activity by inhibiting the binding of NRP1 to at least one NRP1 ligand.

6. The method of item 5, wherein the NRP1 ligand is SEMA3A, VEGF orTGF-β.

7. The method of item 5 or 6, wherein inhibiting the binding of NRP1 toat least one NRP1 ligand comprises administering an anti-NRP1 antibodyor an NRP1 trap, wherein said trap comprises a NRP1 polypeptide or afunctional fragment or variant thereof.

8. The method of item 7, wherein said NRP1 polypeptide corresponds tosoluble NRP1 isoform 2.

9. The method of item 8, wherein said soluble NPR1 isoform 2 comprisesor consists essentially of a polypeptide having an amino acid sequenceas set forth in FIG. 22 without a signal peptide.

10. The method of item 7, wherein said NRP1 polypeptide corresponds tothe extracellular domain of an NRP1 isoform 1 polypeptide.

11. The method of item 10, wherein said NRP1 isoform 1 polypeptide is asset forth in FIG. 26 and wherein said extracellular domain comprisesamino acids 22 to 859 corresponding to the NRP1 polypeptide shown inFIG. 26 (SEQ ID NO:66) .

12. The method of any one of items 7 to 11, wherein said NRP1 trapcomprises an NRP1 polypeptide comprising (i) amino acids 22 to 609 of aNRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) aminoacids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69 (iv)or a functional fragment or functional variant of (i), (ii) or (iii).

13. The method of any one of items 7 to 12, wherein said anti-NRP1antibody inhibits the binding of SEMA3A to NPR-1 but does notsubstantially inhibit the binding of VEGF to NRP1 and wherein said NRP1trap binds to SEMA3A but does not substantially bind to VEGF165 or has areduced binding affinity for VEGF165 compared to SEMA3A bindingaffinity.

14. The method of item 13, wherein said NRP1 trap (i) lacks completelyor partially domain b1 and/or b2 of NRP1; or (ii) comprises at least oneamino acid point mutation which inhibits VEGF binding to NRP1.

15. The method of item 13, wherein said anti-NRP1 antibody does not bindto domain b1 and/or b2 of NRP1.

16. The method of item 14, wherein said point mutation comprises (a) anamino acid substitution or deletion in domain b1 at an amino acidresidue corresponding to tyrosine 297 of an NRP1 amino acid sequence setforth in FIG. 22 or FIG. 26; (b) an amino acid substitution or deletionin domain b1 at an amino acid residue corresponding to aspartic acid 320of an NRP1 amino acid sequence set forth in FIG. 22 or FIG. 26; and/or(c) an amino acid substitution or deletion in domain b1 at an amino acidresidue corresponding to glutamic acid 319 of an NRP1 amino acidsequence set forth in FIG. 22 or FIG. 26.

17. The method of item 16, wherein said point mutation is a Y297Asubstitution; a D320K substitution and/or a E319K substitution.

18. The method of any one of item 7 to 12, wherein said NRP1 trap: (a)comprises domains a1, a2, b1, b2, and c and of said NRP1 polypeptide;(b) comprises domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c)comprises domains a1, a2, and b1 of said NRP1 polypeptide; (d) comprisesdomains al and a2 said NRP1 polypeptide; (f) comprises domain b1 of saidNRP1 polypeptide, wherein said domain b1 comprises at least one pointmutation at an amino acid residue corresponding to (i) tyrosine 297;(ii) aspartic acid 320 and/or (iii) glutamic acid 319, of a NRP1polypeptide comprising an amino acid sequence as set forth in FIG. 26,wherein said at least one mutation reduces or abrogates binding toVEGF165; (g) lacks completely or partially domain c of said NRP1polypeptide; (h) lacks completely or partially domain b1 of said NRP1polypeptide; (i) lacks completely or partially domain b2 of said NRP1polypeptide; (j) lacks domains b1 and b2 of said NRP1 polypeptide; or(k) lacks domains b1, b2 and c of said NRP1 polypeptide.

19. The method of item 18, wherein (i) said domain al comprises orconsists essentially of an amino acids sequence corresponding to aminoacids 27 to 141 of an NRP1 polypeptide as set forth in FIG. 26; (ii)said domain a2 comprises an amino acid sequence corresponding to aminoacids 147 to 265 of an NRP1 polypeptide as set forth in FIG. 26; (iii)said domain b1 comprises an amino acids sequence corresponding to aminoacids 275 to 424 of an NRP1 polypeptide as set forth in FIG. 26; (iv)said domain b2 comprises an amino acids sequence corresponding to aminoacids 431 to 583 of an NRP1 polypeptide as set forth in FIG. 26; and/or(v) said domain c domain comprises an amino acids sequence correspondingto amino acids 645 to 811 of an NRP1 polypeptide as set forth in FIG.26.

20. The method of item 18, wherein (i) said domain al comprises orconsists essentially of an amino acids sequence corresponding to aminoacids 22 to 148 of an NRP1 polypeptide as set forth in FIG. 26; (ii)said domain a2 comprises an amino acid sequence corresponding to aminoacids 149 to 275 of an NRP1 polypeptide as set forth in FIG. 26; (iii)said domain b1 comprises an amino acids sequence corresponding to aminoacids 276 to 428 of an NRP1 polypeptide as set forth in FIG. 26; (iv)said domain b2 comprises an amino acids sequence corresponding to aminoacids 429 to 589 of an NRP1 polypeptide as set forth in FIG. 26; and/or(v) said domain c domain comprises an amino acids sequence correspondingto amino acids 590 to 859 of an NRP1 polypeptide as set forth in FIG.26.

21. The method of item 7, wherein said method comprises inhibiting thebinding of NRP1 to at least one NRP1 ligand by administering a NRP1 trapconsisting essentially of a trap as set forth in Table 1 or a functionalvariant thereof.

22. The method of any one of items 7 to 20, wherein said NRP1 trapfurther comprises a protein purification domain.

23. The method of 22, wherein said purification domain is apolyhistidine tag.

24. The method of any one of items 7 to 20, wherein said NRP1 trapfurther comprises a FC domain.

25. The method of any one of items 22 to 24, wherein said NRP1 trapcomprises a protease or peptidase cleavage site enabling said proteinpurification domain or FC domain to be removed from said NRP1 trap.

26. The method of item 25, wherein said protease or peptidase is a TEVprotease cleavage site.

27. The method of item 26, wherein said TEV protease cleavage sitecomprises the amino acid sequence GSKENLYFQG.

28. The method of item 4, wherein the method comprises reducing NRP1ligand expression or activity, and wherein the NRP1 ligand is SEMA3A.

29. The method of item 28, comprising reducing SEMA3A activity byinhibiting SEMA3A binding to NRP1 by administering an anti-SEMA3Aantibody which binds to the SEMA domain of SEMA3A.

30. The method of 4, wherein said method comprises reducing NRP1expression by administering a NRP1 antisense, shRNA or siRNA.

31. The method of 4, wherein said method comprises reducing SEMA3Aexpression by administering a SEMA3A antisense, shRNA or siRNA.

32. A NRP1 polypeptide trap comprising s a NRP1 polypeptide or afunctional fragment or variant thereof which binds to SEMA3A, VEGF165and/or TGF-β.

33. The NRP1 polypeptide trap of item 32, wherein said NRP1 polypeptidecorresponds to soluble NRP1 isoform 2.

34. The NRP1 polypeptide trap of item 33, wherein said soluble NPR1isoform 2 comprises or consists essentially of a polypeptide having anamino acid sequence as set forth in FIG. 22 without a signal peptide.

35. The NRP1 polypeptide trap of item 34, wherein said NRP1 polypeptidecorresponds to the extracellular domain of an NRP1 isoform 1polypeptide.

36. The NRP1 polypeptide trap of item 35, wherein said NRP1 isoform 1polypeptide is as set forth in FIG. 26 and wherein said extracellulardomain corresponds to amino acids 22 to 859.

37. The NRP1 polypeptide trap of any one of items 32 to 36, wherein saidNRP1 trap comprises an NRP1 polypeptide comprising (i) amino acids 22 to609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) aminoacids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66;(iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ IDNO: 69 (iv) or a functional fragment or functional variant of (i), (ii)or (iii).

38. The NRP1 polypeptide trap of item any one of items 32 to 37, whereinNRP1 trap binds to SEMA3A but does not substantially bind to VEGF165 orhas a reduced binding affinity for VEGF165 as compared to SEMA3A bindingaffinity.

39. The NRP1 polypeptide trap of item 38, wherein said NRP1 trap (i)lacks completely or partially domain b1 and/or b2 of NRP1; or (ii)comprises at least one amino acid point mutation which inhibits VEGFbinding to NRP1.

40. The NRP1 polypeptide trap of item 39, wherein said point mutationcomprises (a) an amino acid substitution or deletion in domain b1 at anamino acid residue corresponding to tyrosine 297 of an NRP1 amino acidsequence set forth in FIG. 22 or FIG. 26; (b) an amino acid substitutionor deletion in domain b1 at an amino acid residue corresponding toaspartic acid 320 of an NRP1 amino acid sequence set forth in FIG. 22 orFIG. 26; and/or (c) an amino acid substitution or deletion in domain b1at an amino acid residue corresponding to glutamic acid 319 of an NRP1amino acid sequence set forth in FIG. 22 or FIG. 26.

41. The NRP1 polypeptide trap of item 40, wherein said mutation point isa Y297A substitution; a D320K substitution and/or a E319K substitution.

42. The NRP1 polypeptide trap of any one of items 32 to 38, wherein saidtrap: (a) comprises domains a1, a2, b1, b2, and c and of said NRP1polypeptide; (b) comprises domains a1, a2, b1 and b2 of said NRP1polypeptide; (c) comprises domains a1, a2, and b1 of said NRP1polypeptide; (d) comprises domains al and a2 said NRP1 polypeptide; (e)comprises domain b1 of said NRP1 polypeptide, wherein said domain b1comprises at least one point mutation at an amino acid residuecorresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii)glutamic acid 319, of a NRP1 polypeptide comprising an amino acidsequence as set forth in FIG. 26, wherein said at least one mutationreduces or abrogates binding to VEGF165; (f) lacks completely orpartially domain c of said NRP1 polypeptide; (g) lacks completely orpartially domain b1 of said NRP1 polypeptide; (h) lacks completely orpartially domain b2 of said NRP1 polypeptide; (i) lacks domains b1 andb2 of said NRP1 polypeptide; or (j) lacks domains b1, b2 and c of saidNRP1 polypeptide.

43. The NRP1 polypeptide trap of item 42, wherein (i) said domain alcomprises or consists essentially of an amino acids sequencecorresponding to amino acids 27 to 141 of an NRP1 polypeptide as setforth in FIG. 26; (ii) said domain a2 comprises an amino acid sequencecorresponding to amino acids 147 to 265 of an NRP1 polypeptide as setforth in FIG. 26; (iii) said domain b1 comprises an amino acids sequencecorresponding to amino acids 275 to 424 of an NRP1 polypeptide as setforth in FIG. 26; (iv) said domain b2 comprises an amino acids sequencecorresponding to amino acids 431 to 583 of an NRP1 polypeptide as setforth in FIG. 26; and/or (v) said domain c domain comprises an aminoacids sequence corresponding to amino acids 645 to 811 of an NRP1polypeptide as set forth in FIG. 26.

44. The NRP1 polypeptide trap of item 42, wherein (i) said domain alcomprises or consists essentially of an amino acids sequencecorresponding to amino acids 22 to 148 of an NRP1 polypeptide as setforth in FIG. 26; (ii) said domain a2 comprises an amino acid sequencecorresponding to amino acids 149 to 275 of an NRP1 polypeptide as setforth in FIG. 26; (iii) said domain b1 comprises an amino acids sequencecorresponding to amino acids 276 to 428 of an NRP1 polypeptide as setforth in FIG. 26; (iv) said domain b2 comprises an amino acids sequencecorresponding to amino acids 429 to 589 of an NRP1 polypeptide as setforth in FIG. 26; and/or (v) said domain c domain comprises an aminoacids sequence corresponding to amino acids 590 to 859 of an NRP1polypeptide as set forth in FIG. 26.

45. The NRP1 polypeptide trap of item 32, wherein said trap consistsessentially of a trap as set forth in Table 1 or a functional variantthereof.

46. The NRP1 polypeptide trap of any one of items 32 to 44, wherein saidtrap further comprises a protein purification domain.

47. The NRP1 polypeptide trap of item 46, wherein said purificationdomain is a polyhistidine tag.

48. The NRP1 polypeptide trap of any one of items 32 to 47, wherein saidNRP1 trap further comprises a FC domain.

49. The NRP1 polypeptide trap of any one of items 46 to 48, wherein saidNRP1 trap comprises a protease or peptidase cleavage site enabling saidprotein purification domain or FC domain to be removed from said NRP1trap.

50. The NRP1 polypeptide trap of item 49, wherein said protease orpeptidase cleavage site is a TEV protease cleavage site.

51. The NRP1 polypeptide trap of item 50, wherein said TEV proteasecleavage site comprises the amino acid sequence GSKENLYFQG.

52. A nucleic acid encoding the NRP1 polypeptide trap of any one ofitems 32-51.

53. An expression vector comprising the nucleic acid of item 52.

54. A host cell comprising the vector of item 53.

55. A composition comprising the NRP1 polypeptide trap of any one ofitems 32 to 51, the nucleic acid of item 52, the vector of item 53 orthe host cell of item 54 and a suitable carrier.

56. The composition of item 55 for (ii) for preventing or treatinginflammation, or (ii) preventing or reducing hyperactivation of innateimmune response.

57. A compound for preventing or treating inflammation, wherein saidcompound: (a) reduces NRP1 expression or activity; and/or (b) reducesNRP1 ligand expression or activity.

58. A compound for preventing or reducing hyperactivation of innateimmune response, wherein said compound: (a) reduces NRP1 expression oractivity; and/or (b) reduces NRP1 ligand expression or activity.

59. The compound of item 57 or 58, wherein said compound is: (i) A antiSEMA3A antibody; (ii) An anti VEGF165 antibody; (iii) A anti NRP1antibody (iv) A NRP1 trap; (v) A SEMA3A antisense, shRNA or siRNA; (vi)A NRP1 antisense, shRNA or siRNA; or (vii) A VEGF antisense, shRNA orsiRNA.

60. The compound item 59, wherein said compound is an NRP1 polypeptidetrap.

61. A composition for treating or preventing inflammation comprising acompound as defined in any one of items 57-60 and a suitable carrier.

62. A composition for preventing or reducing hyperactivation of innateimmune response comprising a compound as defined in of any one of items57-60 and a suitable carrier.

63. Use of the NRP1 polypeptide trap of any one of items 32-51, thenucleic acid of item 52, the vector of item 53, the host cell of item 54the compound of any one of items 57-60 or the composition of any one ofitems 55, 61 and 62 in the manufacture of a medicament for preventing ortreating inflammation.

64. Use of the NRP1 polypeptide trap of any one of items 32-51, thenucleic acid of item 52, the vector of item 53, the host cell of item 54the compound of any one of items 57-60 or the composition of any one ofitems 55, 61 and 62 in the manufacture of a medicament for preventing ortreating inflammation.

65. Use of the NRP1 polypeptide trap of any one of items 32-51, thenucleic acid of item 52, the vector of item 53, the host cell of item 54the compound as defined in any one of items 57-60 or the composition asdefined in any one of items 55, 61 and 62 for preventing or treatinghyperactivation of innate immune response.

66. Use of a the NRP1 polypeptide trap of any one of items 32-51, thenucleic acid of item 52, the vector of item 53, the host cell of item 54the compound as defined in any one of items 57-60 or the composition asdefined in any one of items 55, 61 and 62 for preventing or reducinghyperactivation of innate immune response.

67. The method of any one of items 1-31, wherein said subject suffers oris likely to suffer from septic shock, arthritis, inflammatory boweldisease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabeticretinopathy, age-related macular degeneration (AMD), retinopathy ofprematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS),age-related cognitive decline/Alzheimer's disease or stroke.

68. The method of any one of items 1-31, the NRP1 polypeptide trap ofany one of items 32-51, the nucleic acid of item 52, the vector of item53, the host cell of item 54, a compound as defined in any one of items57-60 or a composition as defined in any one of items 55, 61 and 62wherein said method, NRP1 polypeptide trap, nucleic acid, vector, hostcell, compound, composition or use is for treating or preventing septicshock, arthritis, inflammatory bowel disease (IBD), cutaneous skininflammation, diabetes, uveitis, diabetic retinopathy, age-relatedmacular degeneration (AMD), retinopathy of prematurity, multiplesclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitivedecline/Alzheimer's disease or stroke.

69. The method of any one of items 1-31, the NRP1 polypeptide trap ofany one of items 32-51, the nucleic acid of item 52, the vector of item53, the host cell of item 54, a compound as defined in any one of items57-60 or a composition as defined in any one of items 55, 61 and 62wherein said method, NRP1 polypeptide trap, nucleic acid, vector, hostcell, compound, composition or use is for treating or preventing septicshock or stroke.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In the appended drawings:

FIGS. 1A-1T show that NRP1 identifies a population of microglia that ismobilized secondary to vascular injury. (FIG. 1A). Schematic depictionof the mouse model of oxygen-induced retinopathy (OIR). The first phase(postnatal day 7-12 (P7-P12)), under 75% oxygen, inducesvasoobliteration. The second phase (under room air) from postnatal day12 to 17 (P7-P17) allows to attain maximal pre-retinalneovascularization. (FIGS. 1B, 1E and 1H) show representative FACS plotsof CD11b+/F4-80+/Gr-1⁻ cells (microglia) in retinas collected at P10(FIG. 1B), P14 (FIG. 1E) and P17 (FIG. 1H) from WT OIR and Normoxiccontrol mice. (FIGS. 1C, 1F and 1I) show the fold change in the numberof retinal microglia in Normoxia (N) and OIR at P10 (FIG. 1C), P14 (FIG.1F) and P17 (FIG. 1I). The number of retinal microglia was significantlyincreased in OIR at all points analyzed (FIGS. 1C, 1F and 1I); n=7-8(Normoxia, (N)), n=7-8 (OIR) (total of 28-32 retinas per condition; each“n” comprises 4 retinas). (FIGS. 1D, 1G and 1J) show the fold change inthe number of NRP1 positive MPs at P10 (FIG. 1D), P14 (FIG. 1G) and P17(FIG. 1J). A proportional increase in the number of NRP1-positivemicroglia was observed in OIR retinas (FIGS. 1D, 1G and 1J); n=3-5(Normoxia, (N)), n=3-5 (OIR) (total of 12-20 retinas per condition; each“n” is comprised of 4 retinas). (FIG. 1K). To investigate the role ofMP-resident NRP1, LysM-Cre/Nrp1^(fl/fl) mice which have significantlycompromised NRP1 expression in retinal microglia were generated (n=3(WT), n=4 (LysM-Cre/Nrp1^(fl/fl), total of 12-16 retinas per condition).Left panel shows the % of NPR-1 positive MPs in WT (LysM-Cre/NRP1^(+/+))and mice deficient in NRP1 in their myeloid cells(LysM-Cre/Nrp1^(fl/fl)) as determined by FACS (right panel). (FIGS. 1L,1N and 1P), FACS analysis at P10 (FIG. 1L), P14 (FIG. 1N) and P17 (FIG.1P) to quantify the number of MPs in LysM-Cre/Nrp1^(fl/fl) mice retinasin Normoxia and OIR. (FIGS. 1M, 1O and 1Q) show the fold change in thenumber of MPs in LysM-Cre/Nrp1^(fl/fl) mice retinas in normoxia and OIR.FACS analysis at P10 and P14 during the proliferative phase of OIR (FIG.1L, FIG. 1N) reveals that MP-resident NRP1 is essential for MPinfiltration into the ischemic retina as LysM-Cre/Nrp1^(fl/fl) mice didnot show an increase in numbers of CD11b+/F4-80+/Gr-1⁻ cells in OIR atthese time points (FIG. 1M, FIG. 1O). At P17, MPs infiltrate independentof NRP1 (P, Q). n=7-8 (N), n=7-9 (OIR) (total of 28-36 retinas percondition; each “n” comprises 4 retinas). (FIG. 1R) Summary graph of MPaccumulation in the retina over the course of OIR in WT andLysM-Cre/Nrp1^(fl/fl) mice. (FIG. 1S, FIG. 1T) Representative FACS plotsdepicting that Gr1⁻/CD11b⁺/F4/80⁺ cells express high levels of CX3CR1and intermediate/low levels of CD45. CX3CR1 high and CD45low cellsexpress NRP1 in WT retinas (S) and do not express NRP1 in retinas fromLysM-Cre/Nrp1^(fl/fl) mice (FIG. 1T). Data is expressed as fold changerelative to control±SEM. *P<0.05, **P<0.001,***P>0.0001;

FIGS. 2A-2L show that NRP1⁺ myeloid cells localize to sites ofpathological neovascularization in the retina. Confocal images ofIsolectin B4 (vessel and microglia stain) and NRP1-stained retinalflatmounts at P14 with budding neovascular tufts in WT (FIG. 2A) andLysM-Cre/Nrp1^(fl/fl) mice (FIG. 2G) and at P17 with mature tufts in WT(FIG. 2D) and LysM-Cre/Nrp1^(fl/fl) mice (FIG. 2J). High magnificationimages reveal co-localization of NRP1-positive microglia (IBA1) withboth nascent (FIG. 2B) and mature tufts (FIG. 2E) as confirmed by 3Dreconstruction (FIG. 2C, FIG. 2D) in WT mice. (FIGS. 2C, 2F, 2I, 2L)show 3D reconstruction of tissue. White arrows in (FIG. 2A, right panel)point to sprouting tufts. White arrows in (FIG. 2B, FIG. 2E) point toNRP1⁺ MPs associated with tufts. LysM-Cre/Nrp1^(fl/fl) mice had less MPsand less tufting (FIGS. 2G-2K). For all IHCs, representative images ofthree independent experiments are shown. Scale bars (FIGS. 2A, 2D, 2G,2J): 100 μm, (FIGS. 2B, 2E, 2H, 2K): 50 μm;

FIGS. 3A-3J show that the NRP1 ligand, SEMA3A, is induced in patientssuffering from proliferative diabetic retinopathy. Angiographies,funduscopies, spectral-domain optical coherence tomography (SD-OCT) andthree-dimensional (3D) retinal maps obtained from patients selected forthe study. Control patients had non-vascular pathologies and werecompared to patients with proliferative diabetic retinopathy (PDR).Control ERM patients shows signs of non-diabetes-related retinal damagesuch as (FIG. 3A, FIG. 3B) tractional tension on vasculature (arrow)secondary to (FIG. 3C) fibrotic tissue (white arrow), posterior vitreousdetachment (arrowhead) and macular bulging (angiography and 3D map).Retinas from PDR patients have (FIG. 3E) neovascularization (inset) with(FIG. 3D) highly permeable microvessels as evidenced by leakage offluorescent dye (inset), (FIG. 3F) microaneurysms (inset arrows) and(FIG. 3G) fibrous scar tissue (arrow), indicative of advancedretinopathy. (FIG. 3H) PDR patients show some evidence of macular edema,including cystoid formation (white arrowhead) due to focal coalescenceof extravasated fluid. (FIG. 3I) Vitreous humour analyzed by ELISA showsincreased levels of SEMA3A protein by 5-fold in patients with PDR; n=17for controls and 17 with PDR. (FIG. 3J) Western blot analysis of equalvolumes of vitreous corroborates the increase in SEMA3A (˜125 KDa and 95KDa) in patients with PDR with respect to controls;

FIGS. 4A-4E show that ligands of NRP1 are induced in the retinalganglion cell layer during OIR. (FIG. 4A, FIG. 4B) Retinas from WT andmyeloid deficient NRP1 k.o. mice (LysM-Cre/Nrpr1^(fl/fl) mice) undernormoxic conditions or in OIR were collected between P10 and P17 andanalyzed by RT-qPCR (oligonucleotide used were as disclosed in Example11, Table 2). SEMA3A mRNA (FIG. 4A) expression was induced throughoutOIR in both WT and LysM-Cre/Nrp1fl/fl retinas while VEGF (FIG. 4B) wassignificantly less induced in k.o. mice (LysM-Cre/Nrp1^(fl/fl)) comparedto WT retinas (stars). Data are expressed as a fold change relative torespective normoxic controls for each time point±SEM; n=4-7; *p<0.05,**p<0.01, ***p<0.001. (FIG. 4C) Laser capture micro-dissection (LCM) wasperformed on P14 mice with care being taken to select avascular retinalzones in OIR. (FIG. 4D, FIG. 4E) RT-qPCR on LCM of retinal layers incontrol and OIR avascular zones showed an induction in both SEMA3A (FIG.4D) and VEGF (FIG. 4E) mRNA in the ganglion cell layer (GCL) during OIRretinas compared to normoxic retinas. VEGF was also induced in the innernuclear layer of OIR retinas (FIG. 4E). Data are expressed as a foldchange relative to normoxic GCL±SEM;

FIGS. 5A-5C show that NRP1⁺ MPs do not proliferate in the retina aftervascular injury. Representative FACS histograms of CD11b+/F4-80+/Gr-1⁻cells obtained from retinas (FIG. 5A) and spleens (FIG. 5B) collected atP14 from WT OIR (right panel) and Normoxic (left panel) control miceinjected with BrdU at P13. The number of BrdU⁺ cells was considerablyhigher in spleens but did not change significantly between OIR andNormoxic mice (FIG. 5C). n=4 (Normoxic, N), n=4 (OIR) (total of 16retinas per condition; each “n” is comprised of 4 retinas). Data areexpressed as a percentage of BrdU+ Gr-1⁻/CD11b+/F4-80+ cells±SEM;

FIGS. 6A-6C show that SEMA3A and VEGF are chemo-attractive towardsmacrophages via NRP1. (FIG. 6A, FIG. 6B) Primary macrophages wereisolated from WT or myeloid-deficient NRP1 k.o. mice(LysM-Cre/Nrp1^(fl/fl) mice) and subjected to a transwell migrationassay with vehicle, MCP-1 (100 ng/ml), SEMA3A (100 ng/ml) or VEGF (50ng/ml) added to the lower chamber. Representative images of migratedcells stained with DAPI are shown (FIG. 6A). SEMA3A or VEGF promotedmacrophage migration to similar extents as the positive control MCP-1(FIG. 6B). To ascertain that SEMA3A and VEGF were stimulating macrophagechemotaxis, cells were pre-treated with the selective ROCK inhibitorY-27632 (100 μg/ml) (FIG. 6B) which abolished chemotaxis. Macrophagesfrom LysM-Cre/Nrp1^(fl/fl) mice were unresponsive to SEMA3A or VEGF butresponsive to MCP-1 (FIG. 6C). Data are expressed as a fold changerelative to control (non-treated cells); n=6-22; **p<0.01, ***p<0.001.Scale bars: 100 μm (FIG. 6A);

FIGS. 7A-7E show that Nrp1⁺ macrophages promote microvascular growth inex vivo choroid explants. (FIG. 7A) Quantification and representativeimages of choroid explants isolated from LysM-Cre/Nrp1^(+/+) andLysM-Cre/Nrp1^(fl/fl) mice (n=6; p=0.018). (FIG. 7B, FIG. 7C)Representative images of choroid explants from LysM-Cre/Nrp1^(+/+) (FIG.7B) and LysM-Cre/Nrp1^(fl/fl) (FIG. 7C) mice following chlodronateliposome treatment (to deplete macrophages) and subsequent addition ofexogenous macrophages (Ma). (FIG. 7D, FIG. 7E) Quantification ofchoroidal microvascular sprouting from LysM-Cre/Nrp1^(+/+) (FIG. 7D) andLysM-Cre/Nrp1^(fl/fl) (FIG. 7E) depicted in B and C (n=6, n.s.: notsignificant, *p<0.05, **p<0.01, ***p<0.001);

FIGS. 8A-8F show that deficiency in myeloid-resident NRP1 reducesvascular degeneration and pathological neovascularization inretinopathy. Wild-type, LysMCre/Nrp1^(+/+) and LysM-Cre/Nrp1^(fl/fl)mice were subjected to OIR and retinas collected at P12 and P17,flatmounted and stained with Isolectin B4. LysM-Cre/Nrp1^(fl/fl) micehad less vasoobliteration at P12 (#3 in FIG. 8A, FIG. 8B) and reducedavascular areas (#3 in FIG. 8C, FIG. 8D) and preretinalneovascularization (#3 in FIG. 8E, FIG. 8F) at P17 compared to bothcontrol WT (#1) or control LysMCre/Nrp1^(+/+) mice (#2). Results areexpressed as percentage of avascular or neovascular area versus thewhole retinal area; n=5-19. Scale bars: B&D: 1 mm and F:500 μm.**p<0.01, ***p<0.001;

FIGS. 9A-9C show that therapeutic intravitreal administration of solubleNRP1 reduces MP infiltration and pathological neovascularization inretinopathy. WT mice were subjected to OIR and injected intravitreallyat P12 with soluble recombinant mouse NRP1 (rmNRP1 comprising domainsa1, a2, b1, b2 and c, see also FIGS. 19C and 20X-20Y) as a trap tosequester OIR-induced ligands of NRP1. At P14, FACS analysis revealed adecrease of over 30% in the number of retinal MPs in rmNRP1 injectedretinas (FIG. 9A). Data are expressed as a fold change relative tocontrol (vehicle-injected retinas)±SEM; n=3-4 (total of 12-16 retinasper condition; each “n” comprises 4 retinas). Treatment with rmNRP1efficiently decreased pathological neovascularization at P17 whencompared to vehicle-injected eyes (FIG. 9B, FIG. 9C). Results areexpressed as percentage of neovascular area versus the whole retinalarea; n=11. Scale bars: 500 μm. *p<0.05, **p<0.01;

FIG. 10 is a schematic depiction of the instant findings illustratingthat during ischemic retinopathies such as that of diabetes, avascularzones of the retina, ischemic neurons and neural tissue produces ligandsof NRP1 (SEMA3A and VEGF), which in turn act as potent chemo-attractiveagents for pro-angiogenic microglia. The NRP1⁺ microglia then partake inthe pathogenesis of proliferative retinopathy;

FIGS. 11A-11D show that SEMA3A is upregulated in several organs duringseptic shock. mRNA levels of SEMA3A (left panels) and VEGF (rightpanels) were assessed by qRT-PCR following LPS-induced (15 mg/kg) sepsisin mice. SEMA3A and VEGF mRNA levels were normalized with β-actinexpression and fold changes in mRNA levels were determined at 0, 6, 12and 24 hours following LPS administration. FIG. 11A. Fold change inSEMA3A (left panel) and VEGF (right panel) in mice brain. FIG. 11B. Foldchange in SEMA3A (left panel) and VEGF (right panel) in mice kidneys.FIG. 11C. Fold change in SEMA3A (left panel) and VEGF (right panel) inmice lungs. FIG. 11D. Fold change in SEMA3A (left panel) and VEGF (rightpanel) in mice liver;

FIGS. 12A-12D show cytokines expression following LPS-induced sepsis.mRNA levels of TNF-α and IL-1β were assessed by qRT-PCR followingLPS-induced sepsis (15 mg/kg) in mice. mRNA levels were normalized withβ-actin expression an fold changes in mRNA levels were determined at 0,6, 12 and 24 hours following LPS administration. FIG. 12A. Fold changein TNF-α (left panel) and IL-1β (right panel) in mice brain. FIG. 12B.Fold change in TNF-α (left panel) and IL-1β (right panel) in micekidneys. FIG. 12C. Fold change in TNF-α (left panel) and IL-1β (rightpanel) in mice lungs. FIG. 12D. Fold change in TNF-α (left panel) andIL-1β (right panel) in mice liver;

FIGS. 13A-13C show that SEMA3A induces secretion of pro-inflammatorycytokines in myeloid cells via NRP1. Wild-type and NRP1 knock out(LyzM/NRP1^(fl/fl)) myeloid cells were treated with SEMA3A (100 ng/nml)or vehicle and IL-6 (FIG. 13A), TNF-α (FIG. 13B) and IL-1β (FIG. 13C)protein secretion was analyzed by Cytometric Bead Array (CBA);

FIGS. 14A-14D show that myeloid deficiency in NRP1 reduces production ofinflammatory cytokines during sepsis in vivo. NRP1 knock out mice(LyzM/NRP1^(fl/fl)) and control wild type mice were administered vehicleor LPS (15 mg/kg) to induce sepsis. Brains and livers were collected 6hours post LPS injection and mRNA extracted. TNF-α (FIG. 14A, FIG. 14C)and IL-1β (FIG. 14B, FIG. 14D) expression was analyzed by real-timeRT-PCR and levels normalized with β-actin expression level;

FIGS. 15A-15C show that in vivo inhibition of NRP1 activity preventssepsis-induced barrier function breakdown. Mice were administered withi) vehicle, ii) LPS (15 mg/kg); or iii) LPS (15 mg/kg) and an NRP1 trap(Trap-1, FIGS. 19C and 20X-20Y but without an FC domain, NP_032763, 4ug/0.2 mg/kg, i.v.). Vascular permeability in brain (FIG. 15A), kidney(FIG. 15B) and liver (FIG. 15C) was then assessed using an Evan bluepermeation assay (EBP);

FIGS. 16A-16B show that in vivo inhibition of NRP1 activity protectsagainst sepsis. (FIG. 16A) Survival rate of control mice administeredwith i) a high dose of LPS (i.p., 25/mg/kg); or ii) an NRP1 trap (i.v.,0.2 mg/kg of Trap-1, FIGS. 19C and 20X-20Y but without an FC domain,NP_032763) followed by a high dose of LPS (i.p., 25/mg/kg). (FIG. 16B)Comparison of survival rate between myeloid-resident NRP1 knock out mice(LyzM/NRP1^(fl/fl)) and control mice administered with a high dose ofLPS (i.p., 25/mg/kg);

FIGS. 17A-17B show that administration of NRP1 derived trap or myeloiddeficiency in NRP1 lowers inflammatory cytokine production in septicshock. Wild-type mice were administered i) vehicle (n=3), ii) LPS (15mg/kg, n=3) or iii) LPS and an NRP1 trap (Trap-1, FIGS. 19C and 20X-20Ybut without an FC domain, NP_032763). Mice with NRP1 deficient myeloidcells (LyzM-Cre/Nrp^(fl/fl)) were administered LPS (15 mg/kg, n=3).Brains were collected 6 hours post LPS injection and production of TNF-α(FIG. 17A) and IL-6 (FIG. 17B) was measured;

FIGS. 18A-18E shows that administration of NRP1 derived trap protectsagainst ischemic stroke. Mice were subjected to transient middlecerebral artery occlusion (MCAO) and administered vehicle or NRP1 trapand the size of the infarct (stroke) measured on coronal cerebralsections stained with cresyl violet. The unstained area corresponds tothe damaged area. (FIG. 18A), Coronal cerebral sections of MCAO micetreated with vehicle. (FIG. 18B) Coronal cerebral sections of MCAO micetreated with NRP1 Trap-1 (see FIGS. 19C and 20X-20Y but without an FCdomain, NP_032763). (FIG. 18C) Schematic representation of averageinfarct size in mice treated with vehicle or NRP1 trap following MCAO.(FIG. 18D) Neurological impairment (neuroscore) of mice treated withvehicle or NRP1 trap 1 h after MCAO. (FIG. 18E) Neurological impairment(neuroscore) of mice treated with vehicle or NRP1 trap 24 h after MCAO;

FIGS. 19A-19F show a schematic representation of the NRP1 protein andembodiments of NRP1-traps of the present invention. (FIG. 19A). WT NRP1representation showing SEMA3A binding domain (mainly a1a2 with a smallcontribution of b1 and VEGF binding domain (b1b2). The c-domain is theMEM domain that is thought to contribute to NRP dimerization to otherco-receptors. (FIGS. 19B, 19D-19F) Schematic representations ofhuman-derived NRP1 (FIG. 19C) and mouse-derived NRP1 traps;

FIGS. 20A-20CC show the nucleic acid and protein sequences of the NRP1traps depicted in FIGS. 19B and 19C. (FIGS. 20A-20B) Trap 1/TrappeA-fullNRP1-FC amino acid (SEQ ID NO: 114) and nucleotide (SEQ ID NO: 2)sequences; (FIG. 20C) Trap 2-NRP1-FC-Δc-amino acid sequence (SEQ ID NO:115); (FIG. 20D) Trap 2-NRP1-FC-Δc-nucleotide sequence (SEQ ID NO: 4);(FIG. 20E) Trap 3-NRP1-FC-Δb2c-amino acid sequence (SEQ ID NO: 116);(FIG. 20F). Trap 3-NRP1-FC-Δb2c-nucleotide sequence (SEQ ID NO: 6);(FIG. 20G) Trap 4-NRP1-FC-Δb1 b2c-amino acid sequence (SEQ ID NO: 117);(FIG. 20H) Trap 4-NRP1-FC-Δb1 b2c-nucleotide sequence (SEQ ID NO: 8);(FIGS. 20I-20J) Trap 5/Trap I-NRP1-FCΔc-short-amino acid (SEQ ID NO:118) and nucleotide (SEQ ID NO: 10) sequences; (FIGS. 20K-20L) Trap6/Trap D-NRP1-FCΔb2c-short-amino acid (SEQ ID NO: 119) and nucleotide(SEQ ID NO: 12) sequences; (FIG. 20M) Trap 7/Trap C-NRP1-FCΔb1b2c-short-amino acid (SEQ ID NO: 120) and nucleotide (SEQ ID NO: 14)sequences; (FIGS. 20N-20O) Trap 8/TrapJ-full NRP1-FC-VEGF low-amino acid(SEQ ID NO: 121) and nucleotide (SEQ ID NO: 16) sequences; (FIG. 20P)Trap 9-NRP1-FC-Δc-VEGF low-amino acid sequence (SEQ ID NO: 122); (FIG.20Q) Trap 9-NRP1-FC-Δc-VEGF low-nucleotide sequence (SEQ ID NO: 18);(FIG. 20R) Trap 10-NRP1-FC-Δb2c-VEGF low-amino acid sequence (SEQ IDNO:123); (FIG. 20S) Trap 10-NRP1-FC-Δb2c-VEGF low-nucleotide sequence(SEQ ID NO: 20); (FIGS. 20T-20U) Trap 11/TrapL-NRP1-FC-Δc-VEGFlow-Short-amino acid (SEQ ID NO: 124) and nucleotide (SEQ ID NO:22)sequences; (FIGS. 20V-20W) Trap 12/TrapK-NRP1-FC-Δb2 c-VEGFlow-Short-amino acid (SEQ ID NO:125) and nucleotide (SEQ ID NO:24)sequences. (FIGS. 2OX-20Y) Mouse Trap 1-full Nrp1-mFC amino acid (SEQ IDNO: 126) and nucleotide (SEQ ID NO: 26) sequences. (FIGS. 20Z-20AA)Mouse Trap 2-Nrp1-mFCΔc-short amino acid (SEQ ID NO: 127) and nucleotide(SEQ ID NO: 28) sequences. (FIGS. 20BB-20CC) Mouse Trap3-Nrp1-FCΔb2c-short amino acid (SEQ ID NO: 128) and nucleotide (SEQ IDNO: 30) sequences;

FIG. 21 shows human SEMA3A precursor protein sequence (SEQ ID NO: 31).This sequence is further processed into mature form. Residues 1-20correspond to the signal peptide;

FIG. 22 shows human soluble Neuropilin-1 (NRP1) receptor proteinsequence (e.g., GenBank Acc. No. AAH07737.1-SEQ ID NO: 65). Domains a1,a2, b1, b2 and c are shown. Domain al consist of amino acids 23-148;domain a2 consist of amino acids 149-270; domain b1 consist of aminoacids 271-428; domain b2 consists of amino acids 429-590 and domain cconsists of amino acids 591-609;

FIGS. 23A-23B show that SEMA3A traps accelerate vascular regenerationand reduce pathological angiogenesis in ischemic mice retinas in anoxygen-induced retinopathy model. (FIG. 23A) Schematic depiction of themouse model of oxygen-induced retinopathy (OIR) showing the fourprincipal stages of retinopathy i.e., normoxia, vesselloss/vaso-obliteration, proliferation/neovascularization and neovascular(NV) regression. (FIG. 23B) Mean percentage (%) of avascular area(relative to vehicle) at P17 following intravitreal injection ofhistidine tagged Trap G or Trap M (Trap G-HIS (SEQ ID NO: 38) andTrapM-HIS (SEQ ID NO: 42)). Photographs of representative retinasshowing avascular area are shown for each group. Mean percentage ofneovascular area (relative to vehicle) at P17 following intravitrealinjection of histidine tagged Trap G and Trap M. Photographs ofrepresentative retinas showing neovascular area are shown for eachgroup. *p<0.05, **p<0.01, ***p<0.001. n=8-13 animals/group;

FIGS. 24A-24C show that SEMA3A trap prevents vascular leakage and edemain diabetic retinas. (FIG. 24A). Blood glucose levels of mice priorstreptozotocin (STZ) treatment (week 0) and 3 weeks following STZtreatment (diabetic state). (FIG. 24B). Retinal Evans Blue permeationassay (measured at week 8) on mice retinas injected intravitreally with0.5 ug/ml of Trap G, Trap M or 80 μg (1 ul) of anti-VEGF₁₆₄ antibody(AF-493-NA, R&D) at 6 and 7 weeks following STZ administration. (FIG.24C) Retinal Evans Blue permeation assay (measured at week 14) on miceretinas injected intravitreally with 0.5 ug/ml of Trap G or Trap M oranti-VEGF₁₆₄ antibody (AF-493-NA, Novus Biologicals) at 12 and 13 weekspost STZ treatment. *p<0.05, n=4, from 12 animals;

FIGS. 25A-25B show that NRP1 derived trap (anti SEMA3A and VEGF) reduceschoroidal neovascularization in a model of age-related maculardegeneration (AMD) (FIG. 25A). Schematic representation of the methodused for inducing choroidal neovascularization in mice eyes. (FIG. 25B)Choroidal Neovascularization at day 14 post laser burn (mean perfusedFITC/Lectin area). Mice eyes were injected intravitreally with Trap Gright after laser burn:

FIGS. 26A-26B show an alignment between rat (Access. Nos. EDL96784,NP_659566), human (SEQ ID NO: 68, Accession No. NM003873) and mouse (SEQID NO: 67, Accession No. NP_032763) together with an NRP1 consensussequence (SEQ ID NO: 69). The NRP1 signal domain (amino acids 1-21),SEMA3a binding domains a1 (amino acids 22-148, SEQ ID NO:78), a2 (aminoacids 149-175, SEQ ID NO:79), VEGF binding domains b1 (amino acids276-428, SEQ ID NO:80) and b2 (amino acids 429-589, SEQ ID NO:81),domain c (amino acids 590-859, SEQ ID NO:82), transmembrane domain(amino acids 860-883, SEQ ID NO:77) and cytoplasmic domain (amino acids884-923) are identified; and

FIGS. 27A-27H show protein sequence alignments between exemplary trapsof the present invention shown in FIG. 19 but without any histidine orFC tags. (FIGS. 27A-27D). protein sequence alignment between exemplarytraps but lacking the 6×His tag purification domains (G (SEQ ID NO:100),R (SEQ ID NO:101), Z (SEQ ID NO:102), AB (SEQ ID NO:103), AC (SEQ IDNO:104), O (SEQ ID NO:105), Q (SEQ ID NO:106), M (SEQ ID NO:107), P (SEQID NO:108), N (SEQ ID NO:109), W (SEQIDNO: 110), X (SEQ ID NO: 111) andY (SEQ ID NO: 112)) of the present invention comprising a 6× His tagpurification domain. (FIGS. 27E-27H) protein sequence alignment betweenexemplary traps of the present invention but lacking the FC domain ((A(SEQ ID NO:100), I (SEQ ID NO:105), D (SEQ ID NO:107), C (SEQ IDNO:109), J (SEQ ID NO:101), L (SEQ ID NO:106), K (SEQ ID NO:108), S (SEQID NO:113), U (SEQ ID NO:111), V (SEQ ID NO:112)).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present inventors have identified a subset of mononuclear phagocytes(MPs) that responds to local chemotactic cues such as SEMA3A that areconserved between central neurons, vessels and immune cells. NRP1expressing MP's were shown to enter the site of injury and to contributeto (i) tissue damage and/or (ii) pathological activation of the innateimmune response in models of inflammatory conditions including variousforms of inflammatory, proliferative retinopathies (e.g., proliferativediabetic retinopathy, retinopathy of prematurity and age-related maculardegeneration), septic shock and cerebral ischemia/stroke.

The inventors demonstrated that stressed retinal neurons and neuraltissue have the inherent ability to modulate the local innate immuneresponse via unconventional chemotactic agents. NRP1 on microglia wasfound to be a potent chemoattractive receptor for SEMA3A, and VEGF andinhibition of NRP1 signaling in innate immune cells (e.g., usingNRP1-derived traps or NRP1 or SEMA3A antibodies) resulted in protectionagainst MP's induced inflammation and tissue damage.

Patients suffering from late stage proliferative diabetic retinopathy(PDR) were shown to produce elevated levels of SEMA3A whichcounterintuitively acts as a potent attractant for Neuropilin-1(NRP1)-positive MPs. These pro-angiogenic MPs are selectively recruitedto sites of pathological neovascularization in response to locallyproduced SEMA3A as well as VEGF and TGF-β. Furthermore, SEMA3A was shownto be up-regulated in several organs during septic shock and to inducesecretion of inflammatory cytokines by MP's. Inhibition of NRP1 alsoreduced the production of proinflammatory cytokines in sepsis.

Finally NRP1-positive MPs were shown to play a critical role ininflammatory disease progression. Inhibition/abrogation of NRP1myeloid-dependent activity was shown to protect against neovascularretinal disease (vascular degeneration and pathologicalneovascularization), septic shock and neural damages secondary tocerebral ischemia/stroke.

Together, these findings underscore the role of NRP1-positive MPs andtheir ligands in inflammation (and in particular in neuroinflammation)and demonstrate the therapeutic benefit of inhibiting NRP1 cellsignaling to limit hyperactivation of innate immune response (e.g.,tissue damage at the site of injury through recruitment ofmicroglia/macrophages and/or induction of production and/or secretion ofproinflammatory cytokines, and/or vascular leakage/edema). The presentfindings finds applications in the prevention and treatment of diseasesand conditions characterized by sustained (e.g., chronic, persistent) orexcessive/pathological inflammation involving MP recruitment andproinflammatory cytokines production and secretion such as septic shock,arthritis, inflammatory bowel disease (IBD), cutaneous skininflammation, diabetes, uveitis and neuroinflammatory conditions such asdiabetic retinopathy, age-related macular degeneration (AMD),retinopathy of prematurity, multiple sclerosis, amyotrophic lateralsclerosis (ALS), age-related cognitive decline/Alzheimer's disease andstroke.

Inhibition of NRP1-Mediated Cellular Activity

The present inventors have found that by inhibiting NRP1-dependent cellsignaling (and in particular SEMA3A-mediated cell signaling), it ispossible to protect against (prevent or treat) inflammatory diseases andconditions such as those involving hyperactivation of the innate immuneresponse. In particular, inhibition of NRP1-mediated cell-signalingreduces the unwanted (pathological) recruitment of mononuclearphagocytes (MPs, e.g., microglia, macrophages) and theproduction/secretion of proinflammatory cytokines which contribute totissue damage (e.g., increased vascular degeneration, pathologicneovascularization, cell death or cell damages), inflammation and edema.

Thus, in an aspect, the present invention relates to a method oftreating or preventing inflammation comprising inhibiting NRP1-dependentcell-signaling. In a particular aspect, the inflammation isneuroinflammation.

As used herein, the term “inflammation” means a disease or conditionwhich involves the activation of the innate immune response comprisingi) the recruitment of mononuclear phagocytes (e.g., microglia ormacrophages) expressing the NRP1 receptor at the site of inflammation orinjury; and/or ii) the NRP1 dependent production/secretion ofpro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6). The classicalsigns of acute inflammation are pain, heat, redness, swelling, and lossof function. Inflammation can be classified as either acute or chronic.Acute inflammation is the initial response of the body to harmfulstimuli and is achieved by the increased movement of plasma andleukocytes (especially granulocytes) from the blood into the injuredtissues. A cascade of biochemical events propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolonged(sustained) inflammation, known as chronic inflammation, leads to aprogressive shift in the type of cells present at the site ofinflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process. Non-limitingexamples of inflammatory conditions which may be treated or prevented inaccordance with methods of the present invention include septic shock,arthritis, inflammatory bowel disease (IBD), cutaneous skininflammation, diabetes, uveitis and neuroinflammatory conditions such asdiabetic retinopathy (including proliferative diabetic retinopathy(PDR)), age-related macular degeneration (AMD), retinopathy ofprematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS),age-related cognitive decline/Alzheimer's disease and stroke.

In a particular embodiment the inflammatory disease or condition is nota retinopathy. In another embodiment, the inflammatory disease orcondition is not diabetic retinopathy. In another embodiment, theinflammatory disease or condition is not macular edema. In anotherembodiment, the inflammatory disease or condition is not diabeticmacular edema.

In a related aspect, the present invention concerns a method ofinhibiting hyperactivation (or pathological activation) of the innateimmune response comprising inhibiting NRP1-dependent cell-signaling.Such an hyperactivation of innate immune response, is typicallyassociated with acute or chronic activation of any given cell populationof the immune system (innate and adaptive, e.g., mononuclear cellrecruitment in the organ/tissue) beyond levels required to maintaintissue homeostasis. This is often accompanied by heightened productionof cytokines (e.g., TNF-alpha, IL-6), increased vascular permeability,and may result in compromised tissue function.

In another aspect, the present invention concerns a method of treatingor preventing vascular degeneration comprising inhibiting NRP1-dependentcell-signaling.

In a further aspect, the present invention concerns a method of treatingor preventing pathological neovascularization comprising inhibitingNRP1-dependent cell-signaling.

In another aspect, the present invention concerns a method of treatingor preventing septic shock comprising inhibiting NRP1-dependentcell-signaling.

In a yet another aspect, the present invention concerns a method oftreating or preventing neural damages secondary to cerebralischemia/stroke comprising inhibiting NRP1-dependent cell-signaling.

Because NRP1-mediated cell signaling (e.g., MPs recruitment andproduction/secretion of pro-inflammatory cytokines) depends on thebinding of NRP1 to its ligands (e.g., SEMA3A, VEGF and/or TGF-β),inhibition of NRP1-mediated cellular signaling can be achieved in atleast two ways: i) by targeting the expression or activity of NRP1directly (through the use of NRP1 antibodies, NRP1 derived traps or thelike); or ii) by targeting the expression or activity of one or more ofits ligands (e.g., SEMA3A, VEGF and/or TGF-β).

In embodiments, the above methods comprise preferentially orspecifically inhibiting SEMA3A-mediated cell signalling. “Preferentiallyinhibiting” means that the level of inhibition of SEMA3A-mediated cellsignalling is greater than that of other NRP1 ligands (e.g., VEGF165 andTGF-beta). In certain aspects, methods of the present inventionsubstantially do not reduce or inhibit VEGF (e.g., VEGF165) and/orTGF-beta-mediated cell signalling that occur through the interactionwith NRP1. In embodiments, compounds of the present invention (e.g.,NRP1 traps) “preferentially bind” to one ligand over the others (e.g.,preferentially bind SEMA3A over VEGF). Such preferential interaction maybe determined by measuring the dissociation constant (Kd) for eachligand. In embodiments, interaction for one ligand (e.g., SEMA3A) overthe others (e.g., VEGF) is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 28, 20, 22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400,500, 1000 times greater or more. In embodiments the kD (e.g., in nM) forone ligand is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 28, 20,22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400, 500, 1000times smaller than the kD for one or more of the other ligands (e.g.,VEGF).

In an embodiment, methods of the present invention compriseadministration to a subject likely to suffer from inflammation (e.g.,likely to suffer from an inflammatory disease or condition). In otherembodiment, methods of the present invention comprise administration toa subject diagnosed from inflammation (e.g., likely to suffer from aninflammatory disease or condition). In an embodiment, the subject is amammal, preferably a human.

NRP1 Traps

Inhibition of NRP1-mediated cellular signaling can be achieved usingNRP1 traps of the present invention. As used herein, the terms, “NRP1trap”, or “NRP1 polypeptide trap” encompass naturally occurring solubleNRP1 polypeptide (e.g., such as NRP1 secreted isoform b FIG. 22, SEQ IDNO: 65)), and synthetic (e.g., recombinantly produced) NRP1 polypeptidetraps including any functional soluble fragment of NRP1 (e.g., NRP1isoform 1 or 2) or any functional variant of NRP1 which competes withendogenous NRP1 for ligand binding. In an embodiment, the NRP1 traps ofthe present invention do not exists in nature (i.e., are not naturallyoccurring) but are “derived” from naturally occurring NRP1 polypeptides(i.e. they are synthetic; e.g., NRP1 traps comprising the extracellulardomain of NRP1 isoform 1 or a fragment or variant thereof). NRP1 trapsthe present invention initially comprise a signal peptide at theirN-terminal end (e.g., amino acids 1-21 (SEQ ID NO: 70) of NRP1 shown inFIG. 26 (e.g., SEQ ID NO:69) which is cleaved upon secretion by thecells. Accordingly, NRP1 polypeptide traps of the present invention lackamino acids 1-21 when administered as purified polypeptides or whenprepared as pharmaceutical compositions comprising a purified orsubstantially pure form. Nucleic acids encoding for NRP1 traps of thepresent invention (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 34, 32, 34, 36, 39, 41, 43, 45, 47, etc. See alsoTable 1) comprise a polynucleotide sequence in 5′ which encodes for asignal peptide (first 63 nucleotides encoding for the first 21 aminoacids at the N-terminal end) which will allow the NRP1 trap to besynthesized and secreted by the cells. In a particular embodiment, thesignal peptide corresponds to the first 20 amino acids of the NRP1polypeptide set forth in SEQ ID NO: 65 (FIG. 22) or SEQ ID NO: 69 (FIG.26). NRP1 traps of the present invention encompass functional variantsof corresponding “wild-type” NRP1 polypeptides or fragment thereof(e.g., polymorphic variations naturally found in the population).

NRP1 traps of the present invention may or may not comprise furtherpolypeptide domains (e.g., purification domains). Exemplary trapslacking purification domains and comprising only NRP1-derived sequencesare shown in FIG. 27. Non-limiting examples of NRP1 traps that may beused in accordance with the present invention are given in FIGS. 19B-F,FIG. 20, FIG. 27 and are listed Table 1 below.

TABLE 1 Exemplary NRP1-derived traps which have been prepared inaccordance with the present invention. Trap Description SEQ ID Nos. (aaand nts) Trap 1/A Human, “full” extracellular domain NRP1 SEQ ID NOs: 1,2, (corresponding to amino acids 22 to 856 of NRP1 100 (aa withoutsequences shown on FIG. 26)-FC FC, includes SP) Trap 2 Human, NRP1-FC-Δc(275 aa linker) SEQ ID NOs: 3, 4 Trap 3 Human, NRP1-FC-Δb2c (434 aalinker) SEQ ID NOs: 5, 6 Trap 4 Human, NRP1-FC-Δb1b2c (593 aa linker)SEQ ID NOs: 7, 8 Trap 5/Trap I Human, NRP1-FC-Δc-short SEQ ID NOs: 9,10, 105 (aa, without FC, includes SP) Trap 6/TrapD Human,NRP1-FC-Δb2c-short SEQ ID NOs: 11, 12, 107, (aa, without FC, includesSP) Trap 7/TrapC Human, NRP1-FC-Δb1b2c-short SEQ ID NOs: 13, 14 109,(aa, without FC, includes SP) Trap 8/TrapJ Human, “full” extracellulardomain NRP1-FC-VEGF SEQ ID NOs: 15, 16, low (Y297A mutation) 101 (aa,without FC, includes SP) Trap 9 Human, NRP1-FC-Δc-VEGF low SEQ ID NOs:17, 18 (Y297A mutation, 275 aa linker) Trap 10 Human, NRP1-FC-Δb2c-VEGFlow SEQ ID NOs: 19, 20 (Y297A mutation, 434 aa linker) Trap 11/Trap LHuman, NRP1-FC-Δc-VEGF low-short SEQ ID NOs: 21, 22, (Y297A mutation)106 (aa, without FC, includes SP) Trap 12Trap K Human, NRP1-FC-Δb2c-VEGFlow-short SEQ ID NOs: 23, 24, (Y297A mutation) 108 (aa, without FC,includes SP) mTrap 1 Mouse, “full” extracellular domain NRP1-FC SEQ IDNOs: 25, 26 Amino acids residues 22-856 mTrap 2 Mouse, NRP1-FC-Δc-shortSEQ ID NOs: 27, 28 mTrap 3 Mouse, NRP1-FC-Δb2c-short SEQ ID NOs: 29, 30Trap S Human, NRP1-FC-Δb2-short SEQ ID NOs: 31, 32, 113 (aa without FC,includes SP) Trap U Human, NRP1-FC-Δb2-VEGF low-short SEQ ID NOs: 33,34, (Y297A mutation) 111 (aa, without FC, includes SP) Trap V Human,NRP1-FC-Δb1b2-short SEQ ID NOs: 35, 36, 112 (aa, without FC, includesSP) Trap G Human, “full” extracellular domain NRP1-His SEQ ID NOs: 38,39, 100 (aa without his tag, includes SP) Trap O Human,NRP1-His-Δc-short SEQ ID NOs: 40, 41, 105 (aa without his tag, includesSP) Trap M Human, NRP1-His-Δb2c-short SEQ ID NOs: 42, 43, 107 (aawithout his tag, includes SP) Trap N Human, NRP1-His-Δb1b2c-short SEQ IDNOs: 44, 45, 109 (aa without his tag, includes SP) Trap R Human,NRP1-His-Δc-VEGF low SEQ ID NOs: 46, 47, 101 (aa without his tag) Trap QHuman, NRP1-His-Δc-VEGF low-short SEQ ID NOs: 48, 49, 106 (aa withouthis tag, includes SP) Trap P Human, NRP1-His-Δb2c-VEGF low-short SEQ IDNOs: 50, 51, 108 (aa without his tag, includes SP) Trap W Human, NRP1-His-Δb2 -short SEQ ID NOs: 52, 53, 110 (aa without his tag, includes SP)Trap X Human, NRP1- His-Δb2 - VEGF low-short SEQ ID NOs: 54, 55, 111(aa, without his tag, includes SP) Trap Y Human, NRP1- His-Δb1b2 -shortSEQ ID NOs: 56, 57, 112 (aa, without his tag, includes SP) Trap ABHuman, “full” extracellular domain NRP1-His- SEQ ID NOs: 58, 59, SEMA3Alow (S346A et E348K mutations) 103 (aa, without his tag, includes SP)Trap AC Human, “full” extracellular domain NRP1-His- SEQ ID NOs: 60, 61,VEGF- low (D320K mutation) 104 (aa without his tag, includes SP) Trap ZHuman, “full” extracellular domain NRP1-His, SEQ ID NOs: 62, 63,VEGF165-Low (E319K/D320K mutations) 102 (aa, without his tag, includesSP) Trap 1bis Human, Trap 1 without FC SEQ ID NO: 83, 84 SP: Signalpeptide

Given that NRP1 distinctly regulates the effects of its ligands onsignal transduction and cellular responses, it may be advantageous tospecifically inhibit the binding of one specific ligand to NRP1 but notthat of the others. For example, as shown herein, at early time pointsof retinal disease, where SEMA3A levels are elevated, VEGF levels remainlow and relatively unchanged compared to non-diabetic controls. Also, inseptic shock, SEMA3A was the sole NRP1 ligand which had a long termeffect and stayed up-regulated for more than 24 hours followinginduction of sepsis. Thus, given the differences in expression kineticsfor each ligand and the fact that neutralization of one ligand (e.g.,VEGF) may be ineffective in certain conditions (or be associated withundesired side effects), specific inhibition of one ligand (e.g.,SEMA3A) binding to NRP1, (but not that of the other(s) (e.g., VEGF)) isadvantageous. Thus, in certain aspects of the methods of the presentinvention, inhibition of SEMA3A-mediated cell signaling, is accomplishedby providing NRP1 Traps having greater affinity for SEMA3 than VEGF orto which VEGF (e.g., VEGF165) does not bind or does not bindsubstantially.

Accordingly, in an embodiment, the soluble NRP1 polypeptide orfunctional fragment or variant thereof (NRP1 trap) of the presentinvention binds to all natural ligands of NRP1 (e.g., SEMA3A, VEGF andTGF-beta, e.g., a soluble NRP1 trap comprising the extracellular domain(e.g., amino acids 22-856 or 22-959 of SEQ ID NO: 66 or 69), Trap 1,(SEQ ID NO: 1) or Trap G (SEQ ID NO: 38)-See also, FIGS. 19 and 27 andTable 1). In an embodiment, the NRP1-derived trap of the presentinvention inhibits SEMA3 and VEGF signaling by binding to both SEMA3Aand VEGF.

In another embodiment, the NRP1 trap of the present invention is apolypeptide which binds to SEMA3A but not to VEGF. For example the NRP1trap may comprise the a1 (e.g., SEQ ID NO:71) and/or a2 subdomain(s)(e.g., SEQ ID NO:72) which bind(s) to SEMA3A but not the b1 (e.g., SEQID NO:73) and/or b2 (e.g., SEQ ID NO: 74) subdomain(s) required for VEGFbinding (e.g., Trap M, (SEQ ID NO: 42), Trap N (SEQ ID NO: 44), Trap12/Trap K(SEQ ID NO: 23), Trap 4 (SEQ ID NO:7), Trap 7/C (SEQ ID NO:13), See also, FIGS. 19 and 27 and Table 1). In an embodiment, theNRP1-derived trap comprises domains a1 and a2 corresponding to aminoacids 22 to 275 of the NRP1 amino acid sequence set forth in FIG. 26(e.g., amino acids 22-275 of SEQ ID NO: 66 or SEQ ID NO: 22-275 of SEQID NO: 69). The NRP1 trap may also comprise a mutation (e.g., a deletionor substitution) which abrogates or reduces significantly the binding ofVEGF to NRP1 but not that of SEMA3A to NRP1 (e.g., Trap 8/trap J (SEQ IDNO:15), Trap 9 (SEQ ID NO: 17),Trap 10 (SEQ ID NO: 19), TRAP 11/L (SEQID NO:21), Trap12/K (SEQ ID NO: 23), Trap U (SEQ ID NO: 34), Trap R (SEQID NO: 46), Trap Q (SEQ ID NO: 48), Trap P (SEQ ID NO: 50, Trap X (SEQID NO:54, Tarp AC (SEQ ID NO: 60), TRAP Z (SEQ ID NO: 62) See also,FIGS. 19 and 27 and Table 1). One non-limiting example of such mutationis a substitution at tyrosine 297 in the b1 domain of NRP1 (e.g., Y297A,FIGS. 19B-D, FIG. 27 and Table 1, e.g., Traps 8, 9, 10, 11, 12, V, R, Q,P and X). Other examples of such mutations comprise a substitution atthe glutamic acid at position 319 and at aspartic acid at position 320in NRP1 (e.g., E319K and D320K such as in Trap AC and Z (SEQ ID NOs: 60,62)).

In another embodiment, the NRP1 trap is a soluble NRP1 polypeptide orfunctional fragment or variant thereof which binds to VEGF but not toSEMA3A. For example, the NRP1 trap may comprise the b1 (e.g., SEQ ID NO:73) and/or b2 (e.g., SEQ ID NO: 74) domain(s) to bind to VEGF but notthe al (e.g., SEQ ID NO: 71) and/or a2 (e.g., SEQ ID NO: 72)subdomain(s) which bind to SEMA3A. In an embodiment, the NRP1 trapcomprises domains b1b2 corresponding to amino acids 276 to 589 of theNRP1 amino acid sequence set forth in FIG. 26 (e.g., amino acids 276-589of SEQ ID NO: 66 or 276-289 of SEQ ID NO: 69). In another embodiment,the NRP1 trap may comprise a mutation which reduces or abrogate SEMA3Abinding but not that of VEGF. One non-limiting example of such mutationis a substitution at serine 346 and/or glutamic acid 348 of NRP1 (e.g.,S346A and E348K mutations, such as in trap AB (SEQ ID NO: 58)-See alsoFIGS. 19 and 27).

In an embodiment, the soluble NRP1 polypeptide or functional fragmentthereof comprises or consists of traps as set forth in FIGS. 19B-F, 20,27 and Table 1.

In preferred embodiments, the NRP1 traps of the present invention lackthe transmembrane domain (e.g., corresponding to amino acids residues860 to 883 of the NRP1 polypeptide sequences shown in FIG. 26 (such asSEQ ID NO: 66 and 69)) and cytosolic domain (e.g., corresponding toamino acids residues 884-923 of the NRP1 polypeptide isoform 1 sequencesshown in FIG. 26 (such as SEQ ID NO: 66 and 69)) found in for exampleNRP1 isoform 1. In embodiments, the NRP1 traps of the present inventionlacks completely or partially domain c of NRP1. NRP1 isoform 1 comprisesa larger c domain (see FIG. 26), while that of isoform 2 is shorter(e.g., amino acid sequence VLATEKPTVIDSTIQSGIK (SEQ ID NO: 99) shown inFIG. 22). Particularly, domain c is not essential to SEMA3A and VEGFbinding and thus may be excluded from the NRP1 traps used to inhibitNRP1-dependent cell signaling (or SEMA3A-mediated cell signaling). In anembodiment, the NRP1 trap lacks the c domain corresponding to aminoacids 590 to 859 of the NRP1 amino acid sequence set forth in FIG. 26(e.g., amino acids 590 to 859 of SEQ ID NO: 66 or SEQ ID NO: 69). In anembodiment the NRP1 traps of the present invention lack completely orpartially the c domain of isoform 2 as set forth in FIG. 22 (e.g., SEQID NO: 99). In an embodiment, NRP1 traps of the present inventioncomprise domain c of NRP1 isoform 2. In another embodiment, the NRP1derived trap lacks a portion of domain c corresponding to the aminoacids set forth in SEQ ID NO: 75.

The soluble NRP1 polypeptide or functional fragment or variant thereofof the present invention may comprise one or more additional polypeptidedomain(s) to increase in vivo stability and/or facilitate purification.For example, NRP1 traps of the present invention may include a FC domain(or part thereof such as the human FC domain set forth in SEQ ID NO:37.) or a purification tag (e.g., a 6×-histidine tag). Such additionalpolypeptide domain(s) may be linked directly or indirectly (through alinker) to the soluble NRP1 polypeptide or functional fragment thereof.

The soluble NPR1 polypeptide or functional fragment thereof of thepresent invention may optionally include one or more polypeptidelinkers. Such linkers may be used to link one or more additionalpolypeptide domain(s) to the soluble polypeptide of the presentinvention (e.g., a polypeptide domain which increases the stability ofthe polypeptide in vivo and/or a domain which facilitates purificationof the polypeptide). Linker sequence may optionally include peptidase orprotease cleavage sites which may be used to remove one or morepolypeptide fragments or domains (e.g., removal of purification tagprior to in vivo administration of the soluble NRP1 polypeptides orfunctional fragment thereof). One non-limiting example of a linker ordomain which enables cleavage of the polypeptide and removal of, forexample, polypeptide domain(s) (e.g., 6× his tag purification domain)includes a polypeptide comprising a TEV protease cleavage site (e.g.,GSKENLYFQ'G, SEQ ID NO:76). Many other TEV cleavage sites are known andmany other protease/peptidase cleavage sites are known to the skilledperson and may be introduced in the polypeptides of the presentinvention to optionally remove one or more polypeptide domains orfragments.

Polypeptide linkers may also be used to replace totally or partiallydomains which are normally found in the wild-type NRP1 polypeptide butwhich are absent in the soluble NRP1 polypeptide or functional fragmentthereof of the present invention. For example, in the NRP1 traps of thepresent invention, synthetic linkers may replace totally or partiallydomains a1, a2, b1, b2 and c. The length of the linker may correspond tothe entire length of the domain removed or to a portion of it. Suchlinkers may increase protein folding, stability or binding to NRP1ligands. Non-limiting examples of NRP1 traps comprising linkers areshown in FIGS. 19 and 20 (e.g., Trap 2, Trap 3, Trap 4, Trap 9 and Trap10 listed in Table 1). One non-limiting example of a useful polypeptidelinker is a polyarginine polypeptide. Other linkers are known in the artand may be used in accordance with the present invention.

In an embodiment, the NRP1 trap of the present invention comprises: (i)amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide setforth in FIG. 26 (SEQ ID NO: 69); (ii) amino acids 1 to 583 (preferably22 to 583) of the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69);(iii) amino acids 1 to 424 (preferably 22-424) the NRP1 polypeptide setforth in FIG. 26 (SEQ ID NO: 69); (iv) amino acids 1 to 265 (preferably22 to 265) the NRP1 polypeptide set forth in FIG. 26 (SEQ ID NO: 69);(v) 1 to 430 and 584 to 856 (preferably 22-430 and 584-856) the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vi) amino acids 1 to274 and 584 to 856 (preferably 22-274 and 584 to 856) the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69); (vii) amino acids 1 to430 and 584 (preferably 22 to 430 and 584 to 856) of the NRP1polypeptide set forth in FIG. 26 (SEQ ID NO: 69). In a particularembodiment, the above noted traps comprise one or more mutation toreduce VEGF or SEMA3A binding as described above.

In a related aspect, the present invention provides nucleic acidsencoding the NRP1 traps (e.g., traps listed in Table 1 and shown onFIGS. 19, 20 and 27). Such nucleic acids may be included in anexpression vector for expression in cells. Accordingly, the presentinvention further relates to vectors comprising nucleic acids encodingsoluble NRP1 polypeptide or functional fragments thereof and cellscomprising such expression vectors. Nucleic acids encoding a solubleNRP1 polypeptide or functional fragment thereof (i.e., NRP-derived trap)of the present invention may include a polynucleotide portion encoding asignal sequence (e.g., encoding amino acids 1-21 of SEQ ID NO: 65, 66 or69, or SEQ ID NO: 70) for secretion by the cells. Furthermore, nucleicacids of the present invention include nucleic acids with and without atranslation termination “stop” codon at their 3′ end. The translationtermination stop codon may be provided, for example, by an expressionvector into which the nucleic acids of the present invention may becloned.

As used herein, a “functional fragment” or “functional variant” of NRP1(e.g., a functional fragment of soluble NRP1 polypeptide orpolynucleotide of the present invention such as an NRP1) refers to amolecule which retains substantially the same desired activity as theoriginal molecule but which differs by any modifications, and/or aminoacid/nucleotide substitutions, deletions or additions (e.g., fusion withanother polypeptide). Modifications can occur anywhere including thepolypeptide/polynucleotide backbone (e.g., the amino acid sequence, theamino acid side chains and the amino or carboxy termini). Suchsubstitutions, deletions or additions may involve one or more aminoacids or in the case of polynucleotide, one or more nucleotide. Thesubstitutions are preferably conservative, i.e., an amino acid isreplaced by another amino acid having similar physico-chemicalproperties (size, hydrophobicity, charge/polarity, etc.) as well knownby those of ordinary skill in the art. Functional fragments of thesoluble NRP1 include a fragment or a portion of a soluble NRP1polypeptide (e.g., the al and/or a2 domain(s)) or a fragment or aportion of a homologue or allelic variant of NRP1 which retainsinhibiting activity, i.e., binds to SEMA3A, VEGF and/or TGF-β andinhibits the transduction of NRP1-mediated cellular activity.Non-limiting examples of NRP1-mediated cellular activity include i)vascular hyperpermeability; ii) MPs activation and recruitment; iii)inducement of apoptosis; iv) induction of pro-inflammatory cytokines(e.g., TNF-α, IL-1β) production and/or secretion. In an embodiment, theNRP1 polypeptide is at least 80, 85, 88, 90, 95, 98 or 99% identical tothe polypeptide sequence of FIG. 22 (NRP1 isoform 2, SEQ ID NO: 65) oramino acids 1-859 or 22-859 of the NRP1 isoform 1 set forth in FIG. 26(SEQ ID Nos: 66 and 69). In an embodiment, the NRP1 functional fragmentcomprises subdomains a1, a2, b1, b2 and/c which are/is at least 80, 85,88, 90, 95, 98 or 99% identical to subdomain(s) al (e.g., SEQ ID NO: 71or amino acids 22-148 of SEQ ID NO: 66), a2 (e.g., SEQ ID NO: 72, oramino acids 149-275 of SEQ ID NO: 66), b1 (e.g., SEQ ID NO:73 or aminoacids), b2 (e.g., SEQ ID NO: 74 or amino acids 429-589 of SEQ ID NO:66)and/or c (e.g., SEQ ID NO: 75 or amino acids 590-859 of SEQ ID NO: 66)of NRP1 as depicted in FIG. 22 or 26 (SEQ ID NOs:65 and 66respectively). In an embodiment, the NRP1 is a functional variant whichincludes variations (conservative or non-conservative substitution(s)and/or deletion(s)) in amino acids which are not conserved between rat,mouse and human NRP1 (see FIG. 26 and consensus sequence set forth inSEQ ID NO: 69). Preferably, the NRP1 polypeptide/polynucleotide orfragment thereof is human.

TABLE 2 Non-limiting examples of substitutions in the soluble NRP1polypeptide/NRP1 traps of the present invention. WT Amino acid (withref. to FIG. 26, Exemplary SEQ ID NO: 66) Domain substitution(s) N24 a1Serine E26 a1 Lysine D29 a1 Glycine S35 a1 Asparagine D62 a1 Glutamicacid M68 a1 Isoleucine F90 a1 Isoleucine N96 a1 Glycine H98 a1 ArginineF99 a1 Leucine R100 a1 Tryptophan P110 a1 Serine T153 a2 Alanine S155 a2Threonine S170 a2 Cysteine V177 a2 Isoleucine P196 a2 Glutamine D219 a2Glutamic acid I242 a2 Valine 269 a2 Isoleucine 298 b1 Glycine A303 b1valine N323 b1 Lysine K359 b1 Arginine I360 b1 Valine V362 b1 IsoleucineT371 b1 Serine I372 b1 Leucine P378 b1 Alanine V379 b1 Isoleucine L380b1 Isoleucine V392 b1 Phenylalanine, leucine A393 b1 Glycine P396 b1Proline, serine A409 b1 Valine T410 b1 Serine S469 b2 Threonine A476 b2Serine S479 b2 Proline I481 b2 Threonine I487 b2 Valine E491 b2 Asparticacid 498 b2 Valine G518 b2 Alanine M528 b2 Threonine A553 b2 AlanineP555 b2 Serine, threonine A556 b2 Proline G572 b2 Serine A587 c ValineL599 c Proline D601 c Histidine V634 c Isoleucine N667 c Serine 669 cAlanine K672 c Arginine S674 c Arginine N717 c Serine R741 c HistidineA755 c Valine I756 c Valine S805 c Proline A813 c Threonine P820 cThreonine G835 c deletion E838 c Lysine E854 c Aspartic acid T410 b1Serine S449 b2 Alanine

Antibodies

NRP1 cellular activity can be inhibited by using an agent which blocksNRP1 binding to one or more of its ligands (e.g., SEMA3A, VEGF and/orTGF-β). One example of such agent is an antibody which binds to NRP1 andblocks the binding of NRP1 to SEMA3A, VEGF and/or TGF-β.

Alternatively, inhibition of NRP1-mediated cellular signaling can beachieved by using an agent which blocks the binding of an NRP1 ligand tothe NRP1 polypeptide. Non-limiting examples of such agent includes anantibody which binds to SEMA3A, VEGF or TGF-β and blocks theirrespective binding to NRP1.

In a particular aspect of the present invention, antibodies targetingNRP1 block SEMA3A binding to the receptor but do not substantiallyinterfere with VEGF and/or TGF-β binding to NRP1. In an embodiment, theanti NRP1 antibody binds to the a1a2 domains of the NRP1 polypeptide. Inanother embodiment, the anti NRP1 antibody binds to subdomains a1 or a2of the NRP1 polypeptide.

As noted above, anti SEMA3A antibodies may be used to inhibit (i.e.,reduce completely or partially) NRP1-mediated cellular signaling byblocking SEMA3A binding to NRP1. Useful anti SEMA3A antibodies bind tothe SEMA domain of SEMA3A and block the interaction with NRP1. Inembodiments the anti-SEMA3A antibodies used in accordance with thepresent invention include antibodies binding to SEMA3A polypeptidedomains comprising amino acid residues 252-260, 359-366 or 363-380 ofSEMA3A. SEMA3A antibodies which inhibit the binding of SEMA3A to NRP1are known in art and may be used in accordance with the presentinvention.

As used herein, the expression “anti NRP1 antibody” refers to anantibody that specifically binds to (interacts with) a NRP1 protein anddisplays no substantial binding to other naturally occurring proteinsother than the ones sharing the same antigenic determinants as the NRP1protein. Similarly, the expression “anti SEMA3A antibody”, “anti VEGFantibody” or “anti TGF-β antibody” refers to an antibody thatspecifically binds to (interacts with) a SEMA3A, VEGF or TGF-β proteinrespectively and displays no substantial binding to other naturallyoccurring proteins other than the ones sharing the same antigenicdeterminants as the targeted SEMA3ANEGF/TGF-β protein.

Antibodies that can be used in accordance with the present inventioninclude polyclonal, monoclonal, humanized as well as chimericantibodies. The term antibody or immunoglobulin is used in the broadestsense, and covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodiesand antibody fragments so long as they exhibit the desired biologicalactivity. Antibody fragments comprise a portion of a full lengthantibody, generally an antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, single domain antibodies (e.g., from camelids), nanobodies,shark NAR single domain antibodies, and multispecific antibodies formedfrom antibody fragments. Antibody fragments can also refer to bindingmoieties comprising CDRs or antigen binding domains including, but notlimited to, VH regions (VH, VH-VH), anticalins, PepBodies™,antibody-T-cell epitope fusions (Troybodies) or Peptibodies.

Anti-human NRP1/sem3A/VEGF/TGF-β antibodies have been previouslyprepared and are also commercially available from various sourcesincluding Santa Cruz, AbCam, and Cell Signaling.

In general, techniques for preparing antibodies (including monoclonalantibodies, hybridomas and humanized antibodies when their sequences areknown) and for detecting antigens using antibodies are well known in theart and various protocols are well known and available.

Inhibition of the Expression of NRP1 or NRP1 Ligands

Various approaches are available for decreasing the expression (at themRNA or protein level) of NRP1 or its ligands (e.g., SEMA3A, VEGF orTGF-β) to inhibit NRP1 mediated cell signaling and thus reduceinflammation and hyperactivation of innate immune response (i.e., i)production and/or secretion of pro-inflammatory cytokines; ii)recruitment of mononuclear phagocytes (MPs); iii) vascularhyperpermeabilization; and/or iv) edema, v) neuronal damage, choroidalneovascularization etc.). Non-limiting example includes the use of smallhairpin shRNA (RNAi), antisense, ribozymes, TAL effectors targeting theNRP1, SEMA3A, VEGF or Tgf-β promoter or the like.

Expression in cells of shRNAs, siRNAs, antisense oligonucleotides or thelike can be obtained by delivery of plasmids or through viral (e.g.,lentiviral vector) or bacterial vectors.

Therefore, in alternative embodiments, the present invention providesantisense, shRNA molecules and ribozymes for exogenous administration toeffect the degradation and/or inhibition of the translation of mRNA ofinterest. Preferably, the antisense, shRNA molecules and ribozymestarget human NRP1, SEMA3A, VEGF and/or Tgf-β expression. Examples oftherapeutic antisense oligonucleotide applications include: U.S. Pat.No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No. 5,098,890, issued Mar.24, 1992; U.S. Pat. No. 5,087,617, issued Feb. 11, 1992; U.S. Pat. No.5,166,195 issued Nov. 24, 1992; U.S. Pat. No. 5,004,810, issued Apr. 2,1991; U.S. Pat. No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No.4,806,463, issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15,1994; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423; BioWorldToday, Apr. 29, 1994, p. 3.

Preferably, in antisense molecules, there is a sufficient degree ofcomplementarity to the mRNA of interest to avoid non-specific binding ofthe antisense molecule to non-target sequences under conditions in whichspecific binding is desired, such as under physiological conditions inthe case of in vivo assays or therapeutic treatment or, in the case ofin vitro assays, under conditions in which the assays are conducted. Thetarget mRNA for antisense binding may include not only the informationto encode a protein, but also associated ribonucleotides, which forexample form the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. A method ofscreening for antisense and ribozyme nucleic acids that may be used toprovide such molecules as Shc inhibitors of the invention is disclosedin U.S. Pat. No. 5,932,435.

Antisense molecules (oligonucleotides) of the invention may includethose which contain intersugar backbone linkages such asphosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages, phosphorothioates and those with CH₂—NH—O—CH₂,CH₂—N(CH₃)—O—CH₂ (known as methylene(methylimino) or MMI backbone),CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂ —CH₂ backbones(where phosphodiester is O—P—O—CH₂). Oligonucleotides having morpholinobackbone structures may also be used (U.S. Pat. No. 5,034,506). Inalternative embodiments, antisense oligonucleotides may have a peptidenucleic acid (PNA, sometimes referred to as “protein nucleic acid”)backbone, in which the phosphodiester backbone of the oligonucleotidemay be replaced with a polyamide backbone wherein nucleosidic bases arebound directly or indirectly to aza nitrogen atoms or methylene groupsin the polyamide backbone (Nielsen et al., 1991, Science 254:1497 andU.S. Pat. No. 5,539,082). The phosphodiester bonds may be substitutedwith structures which are chiral and enantiomerically specific. Personsof ordinary skill in the art will be able to select other linkages foruse in practice of the invention.

Oligonucleotides may also include species which include at least onemodified nucleotide base. Thus, purines and pyrimidines other than thosenormally found in nature may be used. Similarly, modifications on thepentofuranosyl portion of the nucleotide subunits may also be effected.Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some specific examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention are OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ orO(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-,S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a reporter group; anintercalator; a group for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide and other substituents having similar properties.One or more pentofuranosyl groups may be replaced by another sugar, by asugar mimic such as cyclobutyl or by another moiety which takes theplace of the sugar.

In some embodiments, the antisense oligonucleotides in accordance withthis invention may comprise from about 5 to about 100 nucleotide units.As will be appreciated, a nucleotide unit is a base-sugar combination(or a combination of analogous structures) suitably bound to an adjacentnucleotide unit through phosphodiester or other bonds forming a backbonestructure.

In a further embodiment, expression of a nucleic acid encoding apolypeptide of interest (e.g., SEMA3A or NRP1), or a fragment thereof,may be inhibited or prevented using RNA interference (RNAi) technology,a type of post-transcriptional gene silencing. RNAi may be used tocreate a pseudo “knockout”, i.e. a system in which the expression of theproduct encoded by a gene or coding region of interest is reduced,resulting in an overall reduction of the activity of the encoded productin a system. As such, RNAi may be performed to target a nucleic acid ofinterest or fragment or variant thereof, to in turn reduce itsexpression and the level of activity of the product which it encodes.Such a system may be used for functional studies of the product, as wellas to treat disorders related to the activity of such a product. RNAi isdescribed in for example published US patent applications 20020173478(Gewirtz; published Nov. 21, 2002) and 20020132788 (Lewis et al.;published Nov. 7, 2002). Reagents and kits for performing RNAi areavailable commercially from for example Ambion Inc. (Austin, Tex., USA)and New England Biolabs Inc. (Beverly, Mass., USA).

The initial agent for RNAi in some systems is a dsRNA moleculecorresponding to a target nucleic acid. The dsRNA (e.g., shRNA) is thenthought to be cleaved into short interfering RNAs (siRNAs) which are21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide3′ overhangs). The enzyme thought to effect this first cleavage step hasbeen referred to as “Dicer” and is categorized as a member of the RNaseIII family of dsRNA-specific ribonucleases. Alternatively, RNAi may beeffected via directly introducing into the cell, or generating withinthe cell by introducing into the cell a suitable precursor (e.g. vectorencoding precursor(s), etc.) of such an siRNA or siRNA-like molecule. AnsiRNA may then associate with other intracellular components to form anRNA-induced silencing complex (RISC). The RISC thus formed maysubsequently target a transcript of interest via base-pairinginteractions between its siRNA component and the target transcript byvirtue of homology, resulting in the cleavage of the target transcriptapproximately 12 nucleotides from the 3′ end of the siRNA. Thus thetarget mRNA is cleaved and the level of protein product it encodes isreduced.

RNAi may be effected by the introduction of suitable in vitrosynthesized siRNA (shRNAs) or siRNA-like molecules into cells. RNAi mayfor example be performed using chemically-synthesized RNA.Alternatively, suitable expression vectors may be used to transcribesuch RNA either in vitro or in vivo. In vitro transcription of sense andantisense strands (encoded by sequences present on the same vector or onseparate vectors) may be effected using for example T7 RNA polymerase,in which case the vector may comprise a suitable coding sequenceoperably-linked to a T7 promoter. The in vitro-transcribed RNA may inembodiments be processed (e.g. using E. coli RNase III) in vitro to asize conducive to RNAi. The sense and antisense transcripts are combinedto form an RNA duplex which is introduced into a target cell ofinterest. Other vectors may be used, which express small hairpin RNAs(shRNAs) which can be processed into siRNA-like molecules. Variousvector-based methods and various methods for introducing such vectorsinto cells, either in vitro or in vivo (e.g. gene therapy) are known inthe art.

Accordingly, in an embodiment expression of a nucleic acid encoding apolypeptide of interest (or a fragment thereof e.g., soluble NRP1, NRP1derived traps, may be inhibited by introducing into or generating withina cell an siRNA or siRNA-like molecule corresponding to a nucleic acidencoding a polypeptide of interest (e.g. SEMA3A or NRP1), or a fragmentthereof, or to an nucleic acid homologous thereto. “siRNA-like molecule”refers to a nucleic acid molecule similar to an siRNA (e.g. in size andstructure) and capable of eliciting siRNA activity, i.e. to effect theRNAi-mediated inhibition of expression. In various embodiments such amethod may entail the direct administration of the siRNA or siRNA-likemolecule into a cell, or use of the vector-based methods describedabove. In an embodiment, the siRNA or siRNA-like molecule is less thanabout 30 nucleotides in length. In a further embodiment, the siRNA orsiRNA-like molecule is about 21-23 nucleotides in length. In anembodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplexportion, each strand having a 2 nucleotide 3′ overhang. In embodiments,the siRNA or siRNA-like molecule is substantially identical to a nucleicacid encoding a polypeptide of interest, or a fragment or variant (or afragment of a variant) thereof. Such a variant is capable of encoding aprotein having activity similar to the polypeptide of interest.

A variety of viral vectors can be used to obtain shRNA/RNAi expressionin cells including adeno-associated viruses (AAVs), adenoviruses, andlentiviruses. With adeno-associated viruses and adenoviruses, thegenomes remain episomal. This is advantageous as insertional mutagenesisis avoided. It is disadvantageous in that the progeny of the cell willlose the virus quickly through cell division unless the cell dividesvery slowly. AAVs differ from adenoviruses in that the viral genes havebeen removed and they have diminished packing capacity. Lentivirusesintegrate into sections of transcriptionally active chromatin and arethus passed on to progeny cells. With this approach there is increasedrisk of insertional mutagenesis; however, the risk can be reduced byusing an integrase-deficient lentivirus.

Pharmaceutical Compositions and Kits

Agents which inhibit NRPI-dependent cell signaling (i.e., NRP1inhibitors) of the present invention can be administered to a humansubject by themselves or in pharmaceutical compositions where they aremixed with suitable carriers or excipient(s) at doses to treat orprevent the targeted disease or condition or to raise the desiredcellular response.

Mixtures of these compounds (e.g., NRP1 trap, antibodies, dominantnegative, small inhibitory peptides or the like) can also beadministered to the subject as a simple mixture or in suitableformulated pharmaceutical compositions. A therapeutically effective dosefurther refers to that amount of the compound or compounds sufficient toresult in the prevention or treatment of the targeted inflammatorydisease or condition (e.g., such as septic shock, arthritis,inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes,uveitis and neuroinflammatory conditions such as diabetic retinopathy,age-related macular degeneration (AMD), retinopathy of prematurity,multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-relatedcognitive decline/Alzheimer's disease) or to provide the desiredcellular or physiological response (e.g., amount sufficient to i) reduceedema, ii) reduce activation/recruitment of mononuclear phagocytes(e.g., microglia or macrophages), iii) reduce production or secretion ofinflammatory cytokines (e.g., IL-1β, TNF-α, IL-6, etc.); iv) reducepathological neovascularization; v) reduce vascular degeneration, etc.).

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, physiological media, and the like that arephysiologically compatible. In embodiments the carrier is suitable forocular administration. In other embodiments the carrier is suitable forsystemic administration. In other embodiments the carrier is suitablefor oral administration.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents, such as for ocular, systemic or oral application, is well knownin the art. Except insofar as any conventional media or agent isincompatible with the compounds of the invention, use thereof in thecompositions of the invention is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

Techniques for formulation and administration of the compounds of theinstant application may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

The present invention also concerns kits or commercial packages for usein the methods of the present invention. Such kits may comprisescompounds of the present invention (e.g., compounds which inhibit NRP1cell signaling, including SEMA3A-mediated cell signaling such as traps,antibodies, shRNA cells, vectors, nucleic acids) optionally withinstructions to use the kit.

Routes of Administration/Formulations

Suitable routes of administration may, for example, include systemic,oral and ocular (eye drops or intraocular injections). Preferred routesof administration comprise eye drops and intraocular injections for eyeconditions, oral for chronic inflammatory conditions and systemic forsepsis and certain neuronal conditions such as stroke. The formulationsmay also be in the form of sustained release formulations.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen, For injection, theagents of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer.

The compounds may be formulated for ocular administration e.g., eyedrops or ocular injections. Formulations for injection may be presentedin unit dosage form, e.g., in ampoules or in multi-dose containers, withan added preservative. The compositions may take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and maycontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Furthermore, one may administer the drug in atargeted drug delivery system, for example, in a liposome coated with acell-specific antibody or other delivery system (e.g., to target forexample a specific tissue (e.g., brain) or cell type (e.g., microglia ormacrophages)). Nanosystems and emulsions are additional well knownexamples of delivery vehicles or carriers for drugs. Another example isthe Encapsulated Cell Therapy (ECT) delivery system from Neurotech's,for eye diseases. ECT is a genetically engineered ocular implant thatenables continuous production of therapeutic proteins to the eye forover 2 years. Additionally, the therapy is reversible by simply removingthe implant. The ECT implant is inserted into the vitreous through asingle incision and sutured in place in a 20-minute outpatient surgicalprocedure.

Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art.

The effective dose of the compound inhibits the cellular signalingfunction of NRP1 sufficiently to reduce or prevent one or morephysiological or cellular responses (e.g., vascular hyperpermeability,blood retinal barrier leakage, edema, MPs activation and/or recruitment,proinflammatory cytokines production and/or secretion,neovascularization, neuronal damage, etc.) or to prevent or treat agiven inflammatory disease or condition, without causing significantadverse effects. Certain compounds which have such activity can beidentified by in vitro assays that determine the dose-dependentinhibition of NRP1-mediated cell signaling inhibitors (e.g., agentswhich directly target the expression or activity of NRP1 or agents whichtargets the expression or activity (e.g., binding) of ligands of NRP1.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellularassays. For example, a dose can be formulated in cellular and animalmodels to achieve a circulating concentration range that includes theIC₅₀ as determined in cellular assays (i a, the concentration of thetest compound which achieves a half-maximal inhibition of the cellularsignaling function of NRP1, usually in response to inflammatorymediators such as Il-1β or other activating stimulus such as hypoxia,ischemia, cellular stress, ER stress, etc.

A therapeutically effective amount refers to that amount of the compoundthat results in amelioration of symptoms in a subject. Similarly, aprophylacticaiiy effective amount refers to the amount necessary toprevent or delay symptoms in a patient (e.g., NRP1-mediated vascularhyperpermeability, spotted and/or blurry vision, pericytes loss, macularedema, retinal swelling, blood retinal barrier leakage, mononuclearphagocytes recruitment, production and secretion of pro-inflammatorycytokines, vascular degeneration, pathological neovascularization,neuronal damage, etc.). Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., determining the maximumtolerated dose (MTD) and the ED (effective dose for 50% maximalresponse). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio between MID andED50. Compounds which exhibit high therapeutic indices are preferred.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition.

Dosage amount and interval may be adjusted individually to providelevels of the active compound which are sufficient to maintain the NRP1modulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data; e.g. the concentration necessary to achieve substantial inhibition ofSEMA3A expression or activity (e.g., binding to NRP1 receptor) Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

Definitions

For clarity, definitions of the following terms in the context of thepresent invention are provided.

As used herein, the term “Neuropilin-1 receptor” or “NRP1” receptorrefers to neuropilin-1 and its isoforms, and allelic/polymorphic forms(e.g., HGNC: 8004; Entrez Gene: 8829; Ensembl: ENSG00000099250; OMIM:602069; and UniProtKB: 014786; GenBank Acc. No. AAH07737.1, FIG. 22, SEQID NO: 65). NRP1 is a non-tyrosine kinase multifunctional receptorhaving the particular ability to bind three structurally dissimilarligands via distinct sites on its extracellular domain. It bindsSEMA3A^(18,19) (for example provoking cytoskeletal collapse) andVEGF_(165,) enhancing binding to VEGFR2 (for example increasing itsangiogenic potential). It also binds to TGF-β. Moreover, genetic studiesshow that NRP1 distinctly regulates the effects of VEGF and SEMA3A onneuronal and vascular development. Hence, depending on the ligand,NRP1-mediated cellular response varies.

The basic structure of neuropilin-1 comprises 5 domains: Threeextracellular domains (a1a2 (CUB), b1b2 (FV/FVIII) and c (MAM)), atransmembrane domain and a cytoplasmic domain (See FIGS. 19A and 22 andSEQ ID NO: 65 and 66 and 68). The a1a2 domain is homologous tocomplement components C1r and C1s (CUB) which generally contain 4cysteine residues forming disulfide bridges. This domain binds SEMA3A.Domains b1b2 (FV/FVIII) binds to VEGF. Amino acid Y297 in subdomain b1is important for binding to VEGF as substitution of Y297 to an alaninesignificantly reduces VEGF binding to NRP1. There exists several splicevariants isoforms and soluble forms of NRP1 which are all encompassed bythe present invention.

“Homology” and “homologous” refers to sequence similarity between twopeptides or two nucleic acid molecules. Homology can be determined bycomparing each position in the aligned sequences. A degree of homologybetween nucleic acid or between amino acid sequences is a function ofthe number of identical or matching nucleotides or amino acids atpositions shared by the sequences. As the term is used herein, a nucleicacid/polynucleotide sequence is “homologous” to another sequence if thetwo sequences are substantially identical and the functional activity ofthe sequences is conserved (as used herein, the term ‘homologous’ doesnot infer evolutionary relatedness). Two nucleic acid sequences areconsidered substantially identical if, when optimally aligned (with gapspermitted), they share at least about 50% sequence similarity oridentity, or if the sequences share defined functional motifs. Inalternative embodiments, sequence similarity in optimally alignedsubstantially identical sequences may be at least 60%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% identical. As used herein, a given percentageof homology between sequences denotes the degree of sequence identity inoptimally aligned sequences. An “unrelated” or “non-homologous” sequenceshares less than 40% identity, though preferably less than about 25%identity, with any of the nucleic acids and polypeptides disclosedherein.

Substantially complementary nucleic acids are nucleic acids in which thecomplement of one molecule is substantially identical to the othermolecule. Two nucleic acid or protein sequences are consideredsubstantially identical if, when optimally aligned, they share at leastabout 70% sequence identity. In alternative embodiments, sequenceidentity may for example be at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%. Optimalalignment of sequences for comparisons of identity may be conductedusing a variety of algorithms, such as the local homology algorithm ofSmith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Natl.Acad. Sci. USA 85: 2444, and the computerised implementations of thesealgorithms (such as GAP, BESTFIT, FASTA and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, Madison, Wis.,U.S.A.). Sequence identity may also be determined using the BLASTalgorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10(using the published default settings). Software for performing BLASTanalysis may be available through the National Center for BiotechnologyInformation. The BLAST algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence that either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighbourhood word scorethreshold. Initial neighbourhood word hits act as seeds for initiatingsearches to find longer HSPs. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction ishalted when the following parameters are met: the cumulative alignmentscore falls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program may use asdefaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoffand Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or0.001 or 0.0001), M=5, N=4, and a comparison of both strands. Onemeasure of the statistical similarity between two sequences using theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. In alternativeembodiments of the invention, nucleotide or amino acid sequences areconsidered substantially identical if the smallest sum probability in acomparison of the test sequences is less than about 1, preferably lessthan about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001.

An alternative indication that two nucleic acid sequences aresubstantially complementary is that the two sequences hybridize to eachother under moderately stringent, or preferably stringent, conditions.Hybridisation to filter-bound sequences under moderately stringentconditions may, for example, be performed in 0.5 M NaHPO4, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1%SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols inMolecular Biology, Vol. 1, Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,hybridization to filter-bound sequences under stringent conditions may,for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C.,and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds),1989, supra). Hybridization conditions may be modified in accordancewith known methods depending on the sequence of interest (see Tijssen,1993, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York). Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH. For example,in an embodiment, the compound of the present invention is anantisense/RNAi or shRNA that hybridizes to an NRP1 or SEMA3A nucleicacid sequence (preferably a human sequence).

As used herein the term “treating” or “treatment” in reference toinflammatory diseases or conditions (e.g., retinopathies, cerebralischemia, stroke, sepsis, ect.) is meant to refer to areduction/improvement in one or more symptoms or pathologicalphysiological responses associated with said disease or condition.Non-limiting examples include edema, swelling, itching, pain, vascularhyperpermeability; blood retinal barrier integrity, increase in SEMA3A,VEGF and/or TGF-beta expression, mononuclear phagocyterecruitment/chemotaxis, production and/or secretion of proinflammatorycytokines, vascular or neuronal degeneration, etc.

As used herein the term “preventing” or “prevention” in reference toinflammatory diseases or conditions is meant to refer to a reduction inthe progression or a delayed onset of at least one symptom associatedwith the disease or condition.

The articles “a,” “an” and “the” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle.

The term “including” and “comprising” are used herein to mean, and reused interchangeably with, the phrases “including but not limited to”and “comprising but not limited to”.

The terms “such as” are used herein to mean, and is used interchangeablywith, the phrase “such as but not limited to”.

The present invention is illustrated in further details by the followingnon-limiting examples.

EXAMPLE 1 Materials and Methods (Examples 2-9 and 12)

Generation of LyzM-cre/Nrpfl/fl mice. C57131/6 wild-type (WT) werepurchased from The Jackson Laboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no.004781) and NRP1 floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchasedfrom The Jackson Laboratory and bread to obtain LyzM-cre/Nrpfl/fl withNRP1-deficient myeloid cells.

O₂-induced retinopathy. Mouse pups (WT or LyzM-Cre (Jackson Laboratory)or LysM-Cre/Nrp1^(fl/fl)) and their fostering mothers (CD1, CharlesRiver) were exposed to 75% O₂ from postnatal day 7 (P7) to day 12 andreturned to room air (52). This model serves as a proxy to human ocularneovascular diseases such as diabetic retinopathy characterized by alate phase of destructive pathological angiogenesis (53, 54). Uponreturn to room air, hypoxia-driven neovascularization (NV) develops fromP14 onwards (26). Eyes were enucleated at different time points and theretinas dissected for FACS analysis or mRNA analysis as described. Inother experiments, dissected retinas were flatmounted and incubatedovernight with fluoresceinated isolectin B4 (1:100) in 1 mM CaCl₂ todetermine extent of avascular area or neovascularization area at P17using ImageJ and the SWIFT-NV method (55).

FACS of digested retinas and spleen. Retinas from WT orLysM-Cre/Nrp1^(fl/fl) mice were homogenized and incubated in a solutionof 750 U/mL DNaseI (Sigma) and 0.5 mg/mL of collagenase D (Roche) for 15min at 37° C. with gentle shaking. Homogenates were then filtered with a70 μm cell strainer and washed in PBS+3% fetal bovine serum. Spleensamples were homogenized and incubated with 1 mg/mL of collagenase D for10 min at 37° C. Homogenates were washed in PBS+3% fetal bovine serumand the pellets were resuspended and incubated in lysis buffer (10 mMKCHO₃; 150 mM NH₄Cl; 0.1 mM EDTA) for 5 min at room temperature. Cellsuspensions (retina or spleen) were incubated with LEAF™ purifiedanti-mouse CD16/32 (Biolegend) for 15 min at room temperature to blockFc receptors. Cells were then incubated for 30 min at room temperaturewith the following antibodies: FITC anti-mouse/human CD11b (Biolegend),PE/CY7 anti-mouse Ly-6G/Ly-6C (Gr-1; Biolegend), Pacific blue™anti-mouse F4/80 (Biolegend), 7AAD (BD Biosciences) andanti-mNeuropilin-1 Allophycocyanin conjugated Rat IgG2A (R&D Systems) orRat IgG2A Isotype Control Allophycocyanin conjugated (R&D Systems).

For analysis of CX3CR1 and CD45 expression, additional extracellularstaining was performed using the above mentioned antibodies supplementedwith Alexa Fluor 700 anti-mouse CD45.2 (Biolegend) and anti-mouse CX3CR1Phycoerythrin conjugated Goat IgG (R&D Systems) or Goat IgG Isotype.Control Phycoerythrin conjugated FACS was performed on a LSRII (BDBiosciences) device and data were analysed using FlowJo™ software(version 7.6.5).

BrdU injections. Wild-type mice subjected to OIR or kept in normoxicconditions were injected intraperitoneally with 5-bromo-2-deoxyuridine(BrdU; Sigma) at the dose of 1 mg/mouse dissolved in PBS at P13.

Analysis of BrdU incorporation. The staining was performed on theretinal cells from P14 WT mice. Samples were obtained as describedabove. Extracellular staining was performed as described above (CD45.2(intermediate/low);Gr-1−;CD11b+,F4/80+;7AAD). Cells were then fixed withCytofix/Cytoperm™ Buffer (BD Biosciences) for 30 min and permeabilisedwith Perm/Wash™ Buffer (BD Biosciences) for 10 min. Next, cells weretreated with 300 ug/mL of DNAse for 1 h at 37° C. and washed withPerm/Wash™. Intracellular staining of BrdU was performed usinganti-BrdU-PE antibodies (Ebioscience) or mouse IgG1 κ Isotype Control PEconjugated (Ebioscience) for 25 min at 4° C. Cells were then washed inPerm/Wash™ and resuspended in PBS+3% fetal bovine serum before FACSanalysis on a LSRII (BD Biosciences).

Vitrectomy. All patients previously diagnosed with PDR were followed andoperated by a single vitreoretinal surgeon (FAR). Control patients wereundergoing surgical treatment for non-vascular pathology (ERM(epiretinal membrane) or MH (macular hole)) by the same surgeon. In anoperating room setting, patients underwent surgery under localretro/peribulbar anesthesia. A 5% povidone-iodine solution was used toclean the periocular skin and topical instillation into the eye andwithin the cul-de-sac was left in place for 5 minutes. Three-port25-gauge transconjunctival pars plana vitrectomy was performed through25-gauge valved cannulas (Alcon). Under microscope visualization using awide-angle viewing system (Resight™, Zeiss), undiluted vitreous wascollected with a 25-gauge vitrector. After vitreous biopsy, the infusionline was opened and vitrectomy and membrane peeling was performed in theusual fashion to treat diabetic vitreous hemorrhage and tractionalretinal detachment. This was followed by panretinal endolaserphotocoagulation, fluid-air exchange, and intravitreal anti-VEGFinjection.

Quantification of SEMA3A protein by ELISA. Vitreous samples were frozenon dry ice and immediately after biopsy and stored at −80°. Samples werecentrifuged at 15000×g for 5 minutes at 4° C. prior to analysis. SEMA3Alevels were quantified in supernatants using enzyme-linked immunosorbentassays (ELISAs) following manufacturer's instructions (USCN Life ScienceInc.).

Assessment of SEMA3A protein levels by Western-blot. Equal volumes ofvitreous fluid (20 uL) from PDR and control patients were assessed bystandard SDS-PAGE technique for the presence of SEMA3A (Abcam).

Real-time PCR analysis. RNA was isolated using the GenElute™ MammalianTotal RNA Miniprep Kit (Sigma) and digested with DNase I to preventamplification of genomic DNA. Reversed transcription was performed usingM-MLV reverse transcriptase (Life Technologies) and gene expressionanalyzed using Sybr™ Green (BioRad) in an ABI Biosystems Real-Time PCRmachine. β-actin was used as a reference gene (see Table 2 in Example 10for details on the sequence of the oligonucleotides used.

Immunohistochemistry. For visualization of pan-retinal vasculature,flatmount retinas were stained with stained with Rhodamine labeledGriffonia (Bandeiraea) Simplicifolia Lectin I (Vector Laboratories,Inc.) in 1 mM CaCl₂ in PBS for retinal vasculature and anti-ratNeuropilin-1 antibody, (goat IgG; R&D Systems) and IBA1 (rabbitpolyclonal; Wako).

Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/flmice were anesthetized with 2% isoflurane in oxygen 2 L/min and theneuthanized by cervical dislocation. Then, a small incision in abdominalskin of mouse was performed. Skin was pulled to each size of the mouseand peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min.Then, the harvested cells were centrifuged for 5 min at 1000 rpm,resuspended in medium (DMEM F12 plus 10% FBS and 1%Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C.under a 5% CO₂ atmosphere the medium was changed and cells were culturedfor the next 24 h in the same conditions before use in cytokine ortranswell migration assay.

Transwell migration assay. Migration assays were performed in 24-wellplates with 8 μm pore inserts. Primary peritoneal macrophages (5×105cells) resuspended in 200 μl of medium (DMEM F12 plus 10% FBS and 1%Streptomycin/Penicillin) were added to the upper chamber. 800 μl ofmedium with or without migratory factors: MCP-1 (100 ng/ml), SEMA3A (100ng/ml), and VEGF₁₆₅ (50 ng/ml) was added to the lower chamber. Cellswere allowed to migrate through the insert membrane overnight at 37° C.under a 5% CO₂ atmosphere. In some experiments, cells were firstpretreated with Y-27632 (Sigma), selective ROCK (Rho-associated coiledcoil forming protein serine/threonine kinase) inhibitor (100 μg/ml) for1 h at 37° C. The inserts were then washed with PBS, and nonmigratingcells were swabbed from the upper surface of the insert membrane withcotton buds. Then the membranes with migrated cells were fixed with 4%paraformaldehyde (PFA) for 20 minutes, washed twice with PBS and mountedon the slide. The cells were stained using mounting medium with DAPI(Vector Laboratories, Inc.). Then, 9 random fields per each membranewere photographed using an inverted fluorescence microscope at 20×magnification and the cells were counted using ImageJ software.

Choroidal explants and microvascular sprouting assay. The ex vivochoroid explants and quantification of microvascular sprouting asdescribed previously(56). Briefly, choroids from LysM-Cre/Nrp1^(+/+) andLysM-Cre/Nrp1^(fl/fl) mice (n=6 for each condition) were dissectedshortly after enucleating eyes. After plating segmented choroids into 24well tissue culture plates and covering with Matrigel™ (BD Biosciences),samples were treated with either EGM™-2 medium, EGM-2 medium with PBSfilled liposome (liposome-PBS), or EGM™-2 medium withDichloromethylenediphosphonic acid disodium salt filled liposome(liposome-Clodronate) (Sigma). The packaging of liposomes was performedaccording to (57). Twelve hours later, liposomes containing passengercompounds were removed from the wells followed by washing with PBS.Macrophages from primary peritoneal macrophage cultures (from eitherLysM-Cre/Nrp1^(+/+) or LysM-Cre/Nrp1^(fl/fl) mice) were added tochoroidal explant cultures to investigate the impact of macrophages onmicrovascular sprouting.

Soluble recombinant NRP1. Wild-type mice subjected to OIR wereintravitreally injected with rmNRP1 trap-1 (FIGS. 19C and 20X-20Y, SEQID NO: 25) from plasmid (29) or R&D Systems at P12.

Recombinant proteins used. Recombinant mouse CCL2/JE/MCP-1 (from E.coli) (R&D Systems) concentration used in vitro 100 ng/ml. Recombinanthuman SEMA3A Fc chimera (from murine myeloma cell line, NS0) (R&DSystems) concentration used in vitro 100 ng/ml. -Recombinant humanVEGF₁₆₅ (PeproTech) concentration used in vitro 50 ng/ml.

Statistical analyses. Data are presented as mean±s.e.m. Student's T-testand ANOVA were used, where appropriate, to compare the different groups;a P<0.05 was considered statistically different. For ELISA, statisticalanalysis was performed using nonparametric Mann-Whitney test (GraphPadPrism).

Study approval: Human samples. We obtained approval of human clinicalprotocol and informed consent form by Maisonneuve-Rosemont Hospital(HMR) ethics committee (Ref. CER: 10059) and recruitment of patients forlocal core vitreal biopsy sampling from patients afflicted with T1DM orT2DM. The entire procedure was performed as an outpatient procedure inthe minor procedure room within the ambulatory clinic from theDepartment of Ophthalmology at Maisonneuve-Rosemont Hospital. Allinstruments were opened and handled in a sterile manner. The studyconforms to the tenets of the declaration Helsinki.

Study approval: Animals. All studies were performed according to theAssociation for Research in Vision and Ophthalmology (ARVO) Statementfor the Use of Animals in Ophthalmic and Vision Research and wereapproved by the Animal Care Committee of the University of Montreal inagreement with the guidelines established by the Canadian Council onAnimal Care. C57131/6 wild-type (WT) were purchased from The JacksonLaboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781) and Neuropilin 1floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchased from The JacksonLaboratory.

TABLE 3 Characteristics of Vitrectomy Patients Db Duration Sample Agetype (years) Retinopathy Analysis C1 74 na na MH WB/ELISA C2 54 na naMMD WB/ELISA C3 72 na na ERM WB/ELISA C4 77 na na ERM WB/ELISA C5 82 nana MH WB/ELISA C6 62 na na ERM ELISA C7 65 na na MH ELISA C8 69 na naERM ELISA C9 75 na na MH/Cataract ELISA C10 77 na na Ret. Det. ELISA C1169 na na ERM ELISA C12 68 na na ERM ELISA C13 81 na na ERM ELISA C14 70na na ERM ELISA C15 65 na na MH ELISA C16 74 na na MH ELISA C17 75 na naMH ELISA PDR1 62 2 13 PDR WB/ELISA PDR2 79 2 33 PDR WB/ELISA PDR3 73 215 PDR WB/ELISA PDR4 74 2 10 PDR WB/ELISA PDR5 54 1 20 PDR WB/ELISA PDR660 2 34 PDR WB/ELISA PDR7 77 2 34 PDR WB/ELISA PDR8 71 2 10 PDR ELISAPDR9 35 — — PDR ELISA PDR10 69 2 40 PDR ELISA PDR11 78 —  5 PDR ELISAPDR12 36 2 — PDR ELISA PDR13 81 1 30 PDR ELISA PDR14 70 2 30 PDR ELISAPDR15 74 — 35 PDR ELISA PDR16 67 2 30 PDR ELISA PDR17 69 2  2 PDR ELISAMH: Macular hole MMD: Myopic Macular Degeneration ERM: EpiretinalMembrane PDR: Proliferative Diabetic Retinopathy Ret. Det.: RetinalDetachement

EXAMPLE 2 NRP1 Identifies a Population of Mononuclear Phagocytes (MPs)that are Mobilized Secondary to Vascular Injury

To determine whether MPs (mononuclear phagocytes) such as microglia ormacrophages partake in the vascular pathogenesis associated withproliferative retinopathies, FACS analysis was first carried-out onwhole mouse retinas to elucidate the kinetics of macrophage/microglialaccumulation throughout the evolution of oxygen-induced retinopathy(OIR, FIG. 1A, 75% oxygen from P7-P12 (postnatal day 7-12) to inducevasoobliteration and room air until P17 to attain maximal pre-retinalneovascularization (26,33)) (FIGS. 1B,E,H). Results revealedsignificantly higher numbers of retinal macrophage/microglial cells(Gr-1−, F4/80+, CD11b+, cells, data not shown) in OIR at all time pointsanalysed including a 36% increase during the vaso-obliterative phase atP10 (P=0.0004) (FIG. 1C), a 63% rise during the neovascular phase at P14(P<0.0001) (FIG. 1F) and a 172% surge during maximal neovascularizationat P17 (P=0.0006) (FIG. 11).

Importantly, at each time point investigated, we observed a proportionalincrease in NRP1-positive MPs in OIR with a rise of 37% at P10(P=0.0240) (FIG. 1D), 61% at P14 (P=0.0196) (FIG. 1G) and 155% at P17(P=0.0058) (FIG. 1J) suggesting that this subpopulation of NRP1-positiveMPs was being recruited to the neuroretina during the progression of thedisease. For all OIR experiments, weights of mouse pups were recorded(data not shown) to ascertain adequate metabolic health (35).

In order to establish the role of MP-resident NRP1 in retinopathy, amyeloid specific knockout of NRP1 was generated by intercrossing Nrp1floxed mice with LysM-Cre mice(36) yielding LysM-Cre/Nrp1^(fl/fl)progeny. The resulting mice showed an ˜80% decrease in NRP1 expressionin retinal MPs when compared to LysM-Cre/Nrp1^(+/+) littermate controls(P=0.0004) (FIG. 1K). Of note, mice tested negative for the rd8 mutationof the Crb1 gene (37). LysM-Cre/Nrp1^(fl/fl) mice did not show anydifference in body weight, size, or open-field activity when comparedwith littermates throughout the period of experimentation (from P1-P17)(data not shown) and had similar numbers of resident retinal microglia(data not shown). Remarkably, deletion of NRP1 on myeloid cells fullyabrogated the entry of macrophages/microglia at P10 and P14 OIR (FIG.1L-O) revealing the critical role for this receptor in MP chemotaxisduring the early stages of ischemic retinal injury. At P17, followingmaximal pathological neovascularization, MP infiltration occurs largelyindependent of NRP1 (FIGS. 1P, Q and R). Consistent with a potentialmicroglial identity, the NRP1-expressing Gr1−/CD11b+/F4/80+ cellsidentified above express high levels of CX3CR1 and intermediate/lowlevels of CD45 (FIG. 1S and data not shown). As expected, inLysM-Cre/Nrp1^(fl/fl) retinas, CD45low/CX3CR1 high MPs are devoid ofNRP1 (FIG. 1T).

EXAMPLE 3 NRP1⁺ Myeloid Cells Localize to Sites of PathologicalNeovascularization in the Retina

Given the pronounced influx of NRP1⁺ macrophage/microglia during OIR,the localization of these cells during the progression of disease wasnext determined. Immunofluorescence on retinal flatmounts revealed thatNRP1-positive macrophage/microglia (co-labelled with IBA1 and NRP1) wereintimately associated with nascent pathological tufts at P14 of OIR(FIG. 2A-C) as well as mature tufts at P17 of OIR (FIG. 2D-F). Whitearrows in FIGS. 2B and 2E point to NRP1-positive MPs associated withpre-retinal tufts. NRP1 was also expressed by endothelial cell on theendothelium of neovascular tufts as previously reported (21). Consistentwith data presented in FIG. 1, LysM-Cre/Nrp1fl/fl mice had lower numbersof macrophage/microglia and less pronounced neovascularization (seebelow for full quantification) (FIG. 2G-K).

EXAMPLE 4 SEMA3A is Elevated in the Vitreous of Patients Suffering fromActive Proliferative Diabetic Retinopathy

To establish the clinical relevance of our findings on the obligate roleof NRP1 in MP chemotaxis in retinopathy, the concentrations of SEMA3Adirectly in the vitreous of patients suffering from active PDR wasdetermined. Seventeen samples of undiluted vitreous were obtained frompatients suffering from PDR and 17 from control patients withnonvascular pathology. Detailed characteristics of patients are includedin Table 1 (Example 1). Control patients (20) presented withnon-vascular pathology and showed signs of non-diabetes-related retinaldamage such as tractional tension on vasculature (FIGS. 3A,B (whitearrow)) secondary to fibrotic tissue and macular bulging (FIG. 3C). Incontrast, all retinas from PDR patients showed signs of disc (FIG. 3D)or pre-retinal neovascularization (FIG. 3F), with highly permeablemicrovessels (leakage of fluorescent dye) (FIGS. 3D,G insets),microaneurisms (FIG. 3D-G) and fibrous scar tissue, indicative ofadvanced retinopathy (FIG. 3G). In addition, patients showed someevidence of macular edema due to compromised vascular barrier function,including cystoid formation (white arrowhead) due to focal coalescenceof extravasated fluid (FIG. 3H).

Consistent with a role in PDR, ELISA-based detection of SEMA3A revealeda 5-fold higher concentrations of the protein in the vitreous humor ofpatients with PDR when compared to vitreous from control patients(P=0.0132) (FIG. 3I). Results were confirmed by Western blot analysis onequal volumes of vitreous where SEMA3A (125 and 95 kDa isoforms)(38, 39)were elevated in patients with PDR (FIG. 3J). Thus, upregulation ofSEMA3A in the vitreous is induced in diabetic ocular pathology.

EXAMPLE 5 NRP1 Ligands are Induced in the Retinal Ganglion Cell LayerDuring OIR

To obtain an accurate kinetic profile of expression of the two prominentligands of NRP1 in proliferative retinopathy, levels of SEMA3A and VEGFmessages in the mouse model of OIR were determined. Real-timequantitative PCR (RT-qPCR) on whole retinas revealed that SEMA3A wasrobustly induced in OIR both during the hyperoxic (vasodegenerative)phase at P10 and the ischemic/neovascular stage from P12 to P17 (FIG.4A). The observed induction occurred in both wild-type andLysM-Cre/Nrp1fl/fl retinas. Conversely, as expected, VEGF transcriptsrose exclusively in the ischemic phase of OIR from P12 to P17 (FIG. 4B).Importantly, VEGF was significantly less induced in LysM-Cre/Nrp1fl/flwhen compared to wild-type retinas (minimally increased at P12(P=0.0451) and ˜55% lower at P14 when compared wild-type OIR (P=0.0003))(FIG. 4B) indicative of a healthier retina.

Next, laser capture micro-dissection (LCM) followed by RT-qPCR wasperformed on retinal layers in avascular zones to pinpoint the source ofSEMA3A and VEGF messages in OIR (FIG. 4C). Both SEMA3A and VEGF whererobustly induced in the ganglion cell layer with VEGF also increasing inthe inner nuclear layer (FIG. 4D, E). Thus, the source of both ligandsis geographically consistent with the localization of retinal MPs (FIG.2).

EXAMPLE 6 Mononuclear Phagocytes (MPs) Do Not Proliferate in the RetinaAfter Vascular Injury

In order to determine if the noted rise in NRP1+ MPs was due to aninflux from systemic circulation or an increase in MP proliferationwithin the retina, the local retinal proliferation of these cells wasinvestigated. Mice were systemically injected with BrdU at P13 (24 hoursprior to sacrifice) and FACS analysis was carried out on retinas (FIG.5A) and spleens (FIG. 5B). Within the retina, Gr1−/CD11b+/F4/80+ MPs didnot show significant proliferation (P=0.4708). Considerably moreproliferation was observed in spleens. No significant difference wasobserved between Normoxia and OIR (FIG. 5C). The lack of proliferationof MPs in the retina suggest that noted accretion NRP1+ MPs duringretinopathy has a systemic origin.

EXAMPLE 7 SEMA3A and VEGF₁₆₅ Mobilize MPs via NRP1

In light of the requirement of NRP1 for myeloid cell mobilization tosites of vascular lesion (FIG. 1) as well as the induction of theprincipal ligands of NRP1 in retinopathy (FIG. 3-4) and the likelysystemic origin of these cells (FIG. 5), the propensity of these cues toprovoke chemotaxis of MPs was determined. Primary macrophage cultureswere isolated from wild-type mice and subjected to a Transwell Boydenchamber migration assay. Both SEMA3A (100 ng/ml) (P<0.0001) and VEGF₁₆₅(50 ng/ml) (P=0.0027) provoked macrophage chemotaxis to similarmagnitudes as positive control MCP-1 (100 ng/ml) (P<0.0001) (FIGS. 6A,B). These data were validated by demonstrating that Y-27632, a selectiveinhibitor ROCK (Rho-associated coiled coil forming proteinserine/threonine kinase) abolished their chemotactic properties. ROCK isdownstream of NRP1 signaling (40) and is known to mediate monocytemigration (41). VEGF migration was partially yet not significantlydiminished suggesting a contribution from alternate receptors such asVEGFR1 as recently reported (33). Consistent with a role for NRP1 inSEMA3A and VEGF-mediated chemotaxis, macrophages fromLysM-Cre/Nrp1^(fl/fl) mice were uniquely responsive to MCP-1 and notmobilized by SEMA3A or VEGF (FIG. 6C).

EXAMPLE 8 NRP1⁺ Macrophages Potentiate Microvascular Sprouting Ex Vivo

To investigate the impact of NRP1 expressing macrophages onmicrovascular angiogenesis, choroid tissue from eitherLysM-Cre/Nrp1^(+/+) mice or LysM-Cre/Nrp1^(fl/fl) mice was isolated andgrew in Matrigel™ to assess microvascular sprouting. Choroids fromLysM-Cre/Nrp1^(fl/fl) mice sprout ˜20% less microvessels when comparedto ones from LysM-Cre/Nrp1^(+/+) mice (P=0.018) (FIG. 7A). Toinvestigate the role of NRP1⁺ macrophages in promoting microvascularsprouting, clodronate-liposomes were used to eliminate endogenousmacrophages from the isolated choroid tissues. In explants from bothLysM-Cre/Nrp1^(fl/fl) and LysM-Cre/Nrp1^(+/+) mice, PBS containingliposomes (i.e. vehicle control) had no impact on vascular sprouting,but clodronate-liposomes reduced microvascular sprouting by ˜60%(P=0.0114 for LysM-Cre/Nrp1^(+/+) choroid and P=0.0007 forLysM-Cre/Nrp1^(fl/fl) choroid) (FIGS. 7B-E). To verify whether NRP1⁺macrophages have a propensity to promote angiogenesis, peritonealmacrophages were extracted from LysM-Cre/Nrp1^(+/+) orLysM-Cre/Nrp1^(fl/fl) mice, and introduced into choroid explant culturesthat had previously been treated with clodronate liposomes and washed.LysM-Cre/Nrp1^(+/+) macrophages robustly potentiated microvascularsprouting by 50-100% when compared to macrophages fromLysM-Cre/Nrp1^(fl/fl) mice(P=0.0068 for LysM-Cre/Nrp1^(+/+) choroid andP=0.0491 for LysM-Cre/Nrp1^(fl/fl) choroid) (FIGS. 7D and E) andindependent of the genotype of the choroidal explant.

EXAMPLE 9 Deficiency in Myeloid-Resident NRP1 Reduces VascularDegeneration and Pathological Neovascularization in Retinopathy

Given the obligate role of NRP1 cell signaling in MP infiltration duringthe early stages of OIR (FIG. 1), the impact of myeloid cell-specificablation of NRP1 on the progression of disease was next determined. Uponexit from 75% O₂ at P12, LysM-Cre/Nrp1fl/fl mice showed significantlylower levels of retinal vasoobliteration when compared to wild-type(P=0.0011) and LysM-Cre/Nrp1+|+ (P<0.0001) controls (FIGS. 8A, B). Thismay be attributed to lower levels of IL-1β present in the retinas ofLysM-Cre/Nrp1fl/fl mice (Data not shown). Importantly, at P17 whenpathological neovascularization peaks (26), deletion of myeloid-residentNRP1 profoundly reduced avascular areas (˜35% when compared to wild-type(P<0.0001) and ˜30% compared to LysM-Cre/Nrp1^(+|+) mice (P=0.0008))(FIGS. 8C, D). In turn, significant reductions in destructivepre-retinal neovascularization associated with ischemic retinopathy wereobserved (˜36% when compared to wild-type (P=0.0008) and ˜34% comparedto LysM-Cre/Nrp1+|+ mice (P=0.0013)) (FIGS. 8E, F).

EXAMPLE 10 Preparation of Soluble SEMA3A Neutralizing Traps

High affinity traps to inhibit/neutralize SEMA3A were generated. Thesetraps were derived from Neuropilin 1 (NRP1) and were optionally coupledto 6×-His tag or FC proteins (see FIGS. 19, 20 and 27, and Table 1).Various variants comprising either the entire NRP1 extracellular domainor functional variants capable of maintaining SEMA3A binding weregenerated. Traps containing a b1 domain (which binds to VEGF) andincluding a neutralizing VEGF165 mutation were generated. The traps wereshown to be highly expressed and secreted in transformed human cells.Simple purification and formulation protocols were developed to producetrap samples for SAR and in vivo efficacies studies to follow.

Methods

Cell culture and material. The human Neuropilin 1 (GenBank™ accessionNM_003873, SEQ ID NO: 66) was acquired from Origene Inc. The Origenclone comprises a conservative mutation at amino acid 140 which changesthe leucine for an isoleucine. The 293T (ATCC) cells were grown inDulbecco's modified Eagle's medium supplemented with 10% fetal calfserum. The pFUSE-hIgG1-Fc1 vector was purchased from InvivoGen Inc.

Cloning. The extracellular domain of Neuropilin-1 (residues 1-856), orportions of it, were PCR amplified from Origene clone RC217035 using thePhusion™ high fidelity polymerase (New England Biolabs) and cloned inthe EcoR1-BgIII of pFUSE-hIgG1-Fc1 in frame with the human FC-1 codingsequence. Constructs coding for the soluble versions of the traps weregenerated by inserting a sequence coding for a TEV protease cleavagesite followed by 6× His residues and a stop codon upstream of the FCcoding portion of the corresponding FC constructs. Additional deletions(b1, b1b2) or VEGF165 binding mutations (e.g., Y297A) were introducedusing the Q5 site directed mutagenesis kit (NEB). All constructssequences were verified by Sanger sequencing (Genome Quebec). Thenucleotides and amino acid sequences of the assembled traps are depictedin FIGS. 20 and 27.

Evaluation of traps' expression in human cells. Constructs coding forthe mouse and human traps were transfected in 293T cells. Cells weregrown for 48 hrs post transfection in FreeStyle™ 293 medium(Invitrogen). Cell lysates were prepared from 293T cells 48 hourspost-transfections. Cells were extensively washed with PBS and lysed inice cold lysis buffer(50 mM HEPES pH7.5, 150 mM NaCL, 1.5 mM MgCl2,1%Triton X-100 and 10% glycerol) supplemented with standard amounts ofprotease inhibitors (AEBSF,TPCK,TLCK, aprotinin, leupeptin, pepstatinand E64, Sigma). Cell lysates were cleared by micro centrifugation(12000 g, 20 minutes). Lysates concentrations were determined bystandard micro BCA (Sigma). Equal amounts of protein were loaded on5-20% PAGE-SDS gradient gels and transfered to PVDF (Amersham). Clearedconditioned media from transfected cells were incubated with eitherProtein A sepharose (Pharmacia) or Talon resin (Clontech) for FC or6×His tag. Resins were washed with PBS and diluted in 2× PAGE-SDS samplebuffer prior to gel separation and transfer. The antibody used inimmunoblottings were the anti-human Neuropilin-1 (Cell signaling), themouse monoclonal anti-6×-HIS (In Vitrogen) and the reporter HRP linkedanti-human, mouse and rabbit IgG (BioRAD). All antibodies were used at a1/2000 dilution. Chemiluminescent signal was captured using a Fujiimaging system after incubation of membranes with ECL (Amersham).

Traps expression and purification. 293-T cells were transfected withplasmids encoding the various traps by either the Polyethylamine (PEI)or the calcium phosphate precipitation standard transfections methods.The next day cells were washed twice with serum free media and fed withserum free complete media (Free style 293 media, InVitrogen).Conditioned medium were collected after 60-72 hrs of growth in serumfree media and cleared from cellular debris by swing bucketcentrifugation (2000 RPM, 20 minutes). FC traps were purified fromconditioned media of transfected 293T cells by passage on Protein A or Gsepharose (Pharmacia) followed by extensive washes with PBS and elutionswith 0.1 M glycine pH 3.0. Elution fractions were neutralisedimmediately by the addition of 1/10 volume 1 M Tris pH 8 and 1/10 volumeof 10× PBS pH 7.4. Soluble 6× HIS tagged traps were purified fromconditioned media of transfected 293T cell by passage on Talon agarose(Clontech) followed by extensive washes with PBS and stepwise imidazoleelutions (Range 10-150 uM typically). Samples of purification fractionsof traps were analysed on 5-15% or 5-20% gradient PAGE-SDS gels. Gelwere stained using the Safely Blue staining kit (InVitrogen).

Sterile formulation of purified traps for in vivo injections.Purifications elution fractions from 40 ml of conditioned media werepooled and diluted to a total volume of 10 ml in PBS . Diluted trapproteins were sterilized by filtration through a 0.2 uM low proteinbinding filter (Progene). Protein solutions were concentrated and bufferexchanged with PBS on sterile PES concentration devices (Pierce, nominalMWCO 30 KD). Sterile concentrated Traps samples (˜30-50 ul) wereanalysed and stained on PAGE-SDS as described above.

EXAMPLE 11 Affinity of Traps for SEMA3A and VEGF

Production of AP-VEGF₁₆₅. the coding sequence of the human VEGF165variant 1 (NM_001025366) was sub-cloned in the pAPtag5 vector(GenHunter), in-frame with an Alkaline Phosphatase domain (AP-VEGF165).HEK293T cells were transfected with the AP-VEGF165 construct using apolyethylenimine (PEI) transfection method. Following the overnighttransfection step, cells were cultured for an additional 60 hr in serumfree media (Invitrogen). The cell media were collected and concentratedon a PES device(Pierce). The concentrated AP-VEGF165 ligand was analysedon PAGE-SDS and quantified using SimplyBlue safe stain (Lifetechnologies).

Sema 3A and AP-VEGF₁₆₅ binding assays. Saturation curves for thedeterminations of KD of binding to SEMA 3A or VEGF165 were obtained asfollow. Wells of high protein binding 96 well plates (Maxisorp, Nunc)were coated with purified traps diluted in PBS and blocked afterwardwith binding buffer (PBS containing 2% casein and 0.05% Tween 20). TheSEMA3A-FC (R&D systems) or AP-VEGF165 ligands were diluted in bindingbuffer over an extensive range of concentrations and added to wells.Following an overnight incubation, wells were washed with PBS containing0.05% tween. Bound SEMA3a-FC was detected using an HRP-linked anti-HumanIgG (Biorad) and ECL substrate (Pierce). Alternatively, bound AP-VEGF165was detected using CPD star substrate (Roche). The Chemiluminescentsignal was acquired on a TECAN reader. Dissociation constant (KD) weredetermined by non-linear curve fitting using the Graph Pad prismsoftware.

The relative affinity of traps of the present invention to SEMA3A andVEGF has been assessed. Traps were prepared as described in Example 10.Schematic representation of traps tested is also provided in FIG. 19.

TABLE 4 Dissociation constant of SEMA3A and VEGF for various Traps SEMA3A-FC VEGF165 SEQ ID NOs: Trap binding (nM) binding (nM) (aa and nts) G0.8  6.75 SEQ ID NOs: 38, 39 O 1.05 N.D. SEQ ID NOs: 40, 41 M 0.95 20.13SEQ ID NOs: 42, 43 N >1000 >250    SEQ ID Nos: 44, 45 R 6.15 N.D. SEQ IDNOs: 46, 47 W 1.14 20.73 SEQ ID NOs: 52, 53 Y >750 N.D. SEQ ID NOs: 56,57 Z 4.44 66.96 SEQ ID NOs: 62, 63 AB N.D. 29.51 SEQ ID NOs: 58, 59 AC 4No binding SEQ ID NOs: 60, 61

Soluble NRP1 traps of the present invention bind more efficiently toSEMA3A than VEGF. Such preference for SEMA3A was found surprising sinceSEMA3A and VEGF are considered to normally have the same generalaffinity for NRP1. Increased affinity for SEMA3A may be advantageous inconditions where SEMA3A inhibition is preferred over inhibition of VEGFand may reduce side effects associated with VEGF inhibition.

EXAMPLE 12 Therapeutic Intravitreal Administration of Soluble NRP1Reduces MP Infiltration and Pathological Neovascularization inRetinopathy

To determine the translational potential of the above findings, asoluble recombinant mouse (rm)NRP1 mTrap 1 polypeptide (FIGS. 19C and20X-20Y comprising domains a1, a2, b1, b2 and c of SEQ ID NO.25) wasnext employed as a trap to sequester OIR-induced ligands of NRP1. Asingle intravitreal injection of rmNRP1 at P12 lead to a 30% reductionat P14 (P=0.0282) in the number of microglia present in retinassubjected to OIR (FIG. 9A). This finding attests to the potency ofsoluble NRP1 (1 μl of 50 μg/ml) to compromise microglial mobilization.Intravitreal administration of soluble NRP1 provoked a significant ˜40%decrease in pathological pre-retinal angiogenesis when compared tovehicle injected controls (P=0.0025) (FIGS. 9B,C). Together, these datasuggest that neutralization of ligands of NRP1 is an effective strategyto reduce destructive neovascularization in retinopathy.

EXAMPLE 13 Materials and Methods for Sepsis Model—Examples 14 to 19

Mouse model of sepsis. Studies were performed according to theregulations from the Canadian Guidelines for the Use of Animals inResearch by the Canadian Council on Animal Care. LPS injections weredelivered intra-peritoneally (i.p) in 6-8 weeks old C57BL/6 mice.

Survival assay. For generation of survival data, mice were challengedwith a single intraperitoneal injection of LPS at 25 mg/kg, in a volumeof nearly 100 ul adjusted to mouse weight. Mice were then monitoreduntil reaching critical limit points defined by the Canadian Council ofAnimal Care.

Measurement of pro-inflammatory cytokines. For assessment ofpro-inflammatory cytokines, mice were challenged i.p. with a singleintraperitoneal injection of LPS at 15 mg/kg and sacrificed at varioustime points up to 24 hours. Tissues (Brain, Liver, Kidney) were removedand mRNA was isolated using the GenElute™ Mammalian Total RNA MiniprepKit (Sigma) and digested with DNase I to prevent amplification ofgenomic DNA. Reversed transcription was performed using M-MLV reversetranscriptase and gene expression analyzed using SybrGreen in an ABIBiosystems Real-Time PCR machine. β-actin was used as a reference gene.

Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/flmice were anesthetized with 2% isoflurane in oxygen 2 L/min and theneuthanized by cervical dislocation. Then, a small incision in abdominalskin of mouse was performed. Skin was pulled to each size of the mouseand peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min.Then, the harvested cells were centrifuged for 5 min at 1000 rpm,resuspended in medium (DMEM F12 plus 10% FBS and 1%Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C.under a 5% CO₂ atmosphere the medium was changed.

Cytometric Bead Array (CBA). CBA was performed according tomanufacturer's guidelines (BD Bioscience). Macrophages were isolatedfrom wild type or LyzMcre/NRP1fl/fl mice and subjected to SEMA3A (100ng/ml) or vehicle for 12 hours and processed by CBA.

Trap and anti-VEGF antibody administration. Mice experimental model ofsepsis were treated with human or mice NRP1 trap-1 (FIGS. 19B, C and20A-20B, 20X-20Y, SEQ ID NO: 25 or SEQ ID NO: 83) or VEGF neutralizingantibody (R&D Systems, AF-493-NA).

Experimental design: 3 mice per group. Groups: 1-Vehicle, 2-LPS, and3-LPS+NRP1 Trap 1-Vehicle: NaCl, 2-LPS: 15 mg/kg; and 3-LPS+NRP1-trap:Mice received i.v. a single injection of 4 ug (in a volume of 100 uL) ofrecombinant mouse NRP1-trap corresponding to 0.2 mg/kg, few minutesafter LPS injection.

Permeability tests. For permeability assays, mice were challenged i.p.with a single intraperitoneal injection of LPS at 15 mg/kg, andsacrificed 24 hrs later for tissue sampling. Changes in liver, kidney,and brain vascular permeability were assessed by quantifying Evans Blue(EB) extravasation in tissue. After 24 hrs, a solution of 10 mg/ml of EBwas injected intravenously (55 mg/kg). Two hours later, mice weresacrificed and perfused through the heart with PBS. Tissues were thenremoved, allowed to dry at room temperature 24 hrs, and dry weights weredetermined. EB was extracted in formamide overnight at 65° C. EB wasthen measured at 620 and 740 nm in spectrophotometer.

Real-time PCR analysis. RNA was isolated using the GenElute™ MammalianTotal RNA Miniprep Kit (Sigma) and digested with DNase I to preventamplification of genomic DNA. Reversed transcription was performed usingM-MLV reverse transcriptase (Life Technologies) and gene expressionanalyzed using SybrGreen (BioRad) in an ABI Biosystems Real-Time PCRmachine. β-actin was used as a reference gene. See Table 3 below fordetails on the sequence of the oligonucleotides used.

TABLE 3 Primer sequences used for RT-PCR analysis Target Primer sequenceSEQ ID NO: β-actin (fwd) GAC GGC CAG GTG ATC ACT ATT G SEQ ID NO: 85β-actin (rev) CCA CAG GAT TCC ATA CCC AAG A SEQ ID NO: 86 SEMA3A (fwd)GCT CCT GCT CCG TAG CCT GC SEQ ID NO: 87 SEMA3A (rev)TCG GCG TTG CTT TCG GTC CC SEQ ID NO: 88 VEGF (fwd)GCC CTG AGT CAA GAG GAC AG SEQ ID NO: 89 VEGF (rev)CTC CTA GGC CCC TCA GAA GT SEQ ID NO: 90 Tnf-α (fwd)CCC TCA CAC TCA GAT CAT CTT CT SEQ ID NO: 91 Tnf-α (rev)GCT ACG TGG GCT ACA G SEQ ID NO: 92 IL-1β (fwd)CTG GTA CAT CAG GAC CTC ACA SEQ ID NO: 93 IL-1β (rev)GAG CTC CTT AAC ATG CCC TG SEQ ID NO: 94 IL-6 (fwd)AGA CAA AGC CAG AGT CCT TCA GAG A SEQ ID NO: 095 IL-6 (Rev)GCC ACT CCT TCT GTG ACT CGA GC SEQ ID NO: 96

EXAMPLE 14 Semaphorin 3A is Upregulated in Several Organs During SepticShock

Given the link between SEMA3A, NRP1 and the innate immune response inOIR (as demonstrated in Examples 2-9 above), the implication of theNRP1-dependent cellular response in general systemic inflammation wasnext assessed. This was first explored by determining the kinetics ofSEMA3A expression during septic shock.

LPS was administrated (15 mg/kg) to 6-8 weeks old C57BL/6 mice (n=5) andmice were sacrificed at 0, 4, 8, 12 and 24 hours following LPSadministration. Key organs such as brain, kidney, lung and liver werecollected and mRNA isolated. Levels of SEMA3A mRNA were robustly inducedin all organs analyzed as soon as 6 hours after LPS injection andpersisted for 24 hours (FIG. 11A-D). Similarly, expression levels ofanother NRP1 ligand, VEGF, were also profoundly increased in kidney(FIG. 11B), lung (FIG. 11C) and liver (FIG. 11D) within the first 6hours of septic shock. Increases in classical pro-inflammatory cytokinesTNF-α and IL1-β rose at 6 hours post LPS administration and diminishedsimilarly to VEGF mRNA (FIG. 12). Hence, of all investigated mediatorsof inflammation, SEMA3A had a long-term kinetic profile and stayedelevated for at least 24 hours following induction of sepsis. Thisparticular expression profile for SEMA3A suggests that its contributionto septic shock may be long lasting when compared to other cytokines.

EXAMPLE 15 SEMA3A Induces Secretion of Pro-Inflammatory Cytokines inMyeloid Cells via NRP1

Given the contribution of monocytes and myeloid cells to the acuteinflammatory response and the presence of NRP1 on myeloid cells, thecontribution of SEMA3A and myeloid-resident NRP1 in the production ofinflammatory cytokines was determined.

Isolated macrophages were exposed to SEMA3A (100 ng/ml) or vehicle andthe production of cytokines was analyzed by Cytometric Bead Array (CBA).Results presented in FIG. 13 indicate that SEMA3A can induce theproduction/secretion of pro-inflammatory cytokines, known to contributeto septic shock such as IL-6 (FIG. 13A) and TNF-α (FIG. 13B). Ofparticular importance, a specific knockout of NRP1 (LyzM/NRP1^(fl/fl))in myeloid cells abrogated SEMA3A-induced production of IL-6 and TNF-α.Notably, vehicle-treated control LyzM/NRP1^(fl/fl) macrophages showedlesser production of IL-6, TNF-α and IL-1β then wild-type controls,highlighting the role of myeloid-resident NRP1 in sepsis-inducedinflammation.

EXAMPLE 16 Deficiency in Myeloid-Resident NRP1 Reduces Production ofPro-Inflammatory Cytokines In Vivo in Sepsis

Because myeloid-resident NRP1 was important for the release ofpro-inflammatory cytokines such as IL-6 and TNF-α in vitro, itscontribution was next explored in vivo. LyzM/NRP1^(fl/fl) and controlwild-type mice were administered vehicle or LPS (15 mg/kg) and brainsand livers were collected 6 hours post LPS injection. Real-time PCRanalysis of TNF-α (FIGS. 14A,C) and IL-1b (FIG. 14B,D) levels revealed arobust drop in these cytokines in LyzM/NRP1^(fl/fl). These resultsunderscore the profound contribution of NRP1 and its ligands to thedevelopment of sepsis in vivo.

EXAMPLE 17 Inhibition of NRP1 Signalling Prevents Sepsis-Induced BarrierFunction Breakdown

One of the pathological features of severe septic shock though tocontribute to organ failure is the compromise of blood barriers (bloodand air in lung, blood and urine in the kidney, blood and bile in liver,and humoral molecules in the brain). Given a role for SEMA3A in thebreakdown of the blood retinal barrier (46) and the present novel dataon the expression of SEMA3A during sepsis, the effect of neutralizingSEMA3A with a trap derived from the extracellular domain of human NRP1was assessed (Trap-1, without FC, FIG. 19B, SEQ ID NO:83). Using anEvans Blue Permeation (EBP) assay, we found that in all organs studiednamely brain (FIG. 15A), kidney (FIG. 15B) and liver (FIG. 15C), apronounced reduction in LPS-induced barrier function breakdown wasobserved when mice were treated with 4 ug of NRP1 derived trap (0.2mg/kg, i.v.). These results strongly suggest that traps of soluble NRP1and their derivatives are compelling therapeutic agents to countersepsis.

EXAMPLE 18 NRP1-Derived Trap Protects Against Sepsis

To determine the therapeutic benefits of neutralization of NRP1 ligandsor NRP1 inhibition during sepsis, survival studies were performed. Ahigh dose of LPS (25 mg/kg) was administered to mice. Mice were thenmonitored, and ethically sacrificed, when appropriate endpoints wereachieved. In the second group, mice were injected i.v. with 4 ug ofrecombinant Trap-1 without FC (0.2 mg/kg, FIGS. 19B and 20A-20B, SEQ IDNO: 83) followed by LPS intraperitoneal injection. In the control group,5/5 mice (100%) died within first 30 hrs (FIG. 16A) following LPSinjection. Conversely, all mice treated with the trap were still aliveafter 30 hours and showed significant improved survival rate after 60hours (3/5). Mortality was thus reduced from 100% (in the control group)after 30 hours to 40% (FIG. 16A) after 60 hours. Furthermore, 40% ofTrap treated-mice remained alive 80 hours following LPS injection. Thus,survival time was at least doubled in 60% of the case and almost tripledin 40% of the case when cell signaling through NRP1 was inhibited.

Similar results were obtained with mice harboring a specific knock outof NPR1 in myeloid cells (FIG. 16B). Absence of NRP1 in myeloid cellsincreased survival time and reduced sepsis-induced mortality (3/5) from100% to 40% (FIG. 16B) after 30 hours and from 100% to 40% after 60hours. Also, 40% of NRP1 K.O. mice remained alive 80 hours following LPSinjection.

Taken together, these results highlight the therapeutic value ofinhibiting NRP1-dependent cell signaling in sepsis treatment.

EXAMPLE 19 NRP1-Derived Trap Lowers Production of Inflammatory Cytokinesin Septic Shock

Given the therapeutic benefit of NRP1-trap on survival rates in septicshock, the impact of neutralization of NRP1 ligands on production ofinflammatory cytokines during septic shock was next determined.Wild-type mice were administered i) vehicle (n=3); ii) LPS (15 mg/kg)(n=3) or iii) LPS and NRP1 mouse Trap 1 (without FC, FIG. 19C SEQ ID NO:25, but without FC region) and brains were collected 6 hours post LPSinjection. Injection of NRP1 trap-1 profoundly reduced production ofTNF-α (FIG. 17A) and IL-6 (FIG. 17B). Similarly, mice with NRP1deficient myeloid cells (LyzM-Cre/Nrp^(fl/fl)) (n=3) producedconsiderably less TNF-α and IL-6, underscoring the contribution of thiscellular pathway to the progression of septic shock.

EXAMPLE 20 Materials and Method for the Cerebral Ischemia/Stroke ModelDescribed in Example 21

The mice used in this study were 2- to 3-month old male C57Bl/6 mice(22-28 g).

MCAO model. MCAO mouse model was performed using the intraluminal suturetechnique described by Rousselet et al. (66). Briefly, mice wereanesthetized in a chamber with 3% isoflurane in oxygen (1 L/min) andanalgesized with buprenorphine (0.1 mg/kg body weight subcutaneously).Anesthesia was maintained during the operation using 1.5% isoflurane inoxygen provided via a face mask. The rectal temperature was recorded andkept stable at 37±0.5° C. with a heating pad. After a midline incisionat the neck, the right carotid bifurcation was exposed and the commoncarotid artery (CCA) was temporarily occluded using 5-0 silk suture. Thebifurcation of the right internal common carotid artery (ICA) andexternal common carotid artery (ECA) was separated. A permanent suturewas placed around the ECA, as distally as possible, and anothertemporary suture slightly tight was placed on the ECA distal to thebifurcation. The right ICA was temporarily occluded with 5-0 silk sutureto avoid bleeding. Then, a small hole in the ECA was cut betweenpermanent and temporary sutures through which a 12 mm-long 6-0silicon-coated (about 9-10 mm was coated with silicon) monofilamentsuture was introduced. The filament was advanced from the ECA into thelumen of the ICA until it blocked the origin of the middle cerebralartery (MCA) in the circle of Willis. Sham animals were obtained byinserting the monofilament into the CCA, but without advancing it to theMCA. The suture on the ECA was tightly tied to fix the monofilament inposition. Thirty minutes after MCAO, the monofilament was completelyremoved to allow reperfusion. The temporary suture on the CCA was alsoremoved to allow blood recirculation. After the wound was closed, 1 mlof saline solution was injected subcutaneously to avoid postsurgicaldehydration. The mouse was placed in a cage and kept on the heating padfor 1 h. Meantime, when the mouse was fully awake from anesthesia, itwas checked for some basic motor deficits (circling while walking andbending while hold by tail; indicators of the success of the operation)and NRP1-Trap-1 without FC (FIG. 19B SEQ ID NO:83) at the dose of 0.4 ugin 125 ul of PBS was administered to the tail vein (about 15 min afterreperfusion had been started). Control animals operated in the same wayas NRP1-treated animals received, after MCAO, a vehicle (PBS). Becausepost-surgical weight loss is generally observed, mashed food was placedin a Petri dish to encourage eating.

Determination of infarct volume. Following neurological evaluation (seesection below) performed 24 h after MCAO the animals were deeplyanesthetized with 3% isoflurane in oxygen (1 L/min) and decapitated. Thebrains were immediately isolated and transferred into isopentane cooledon dry ice and then stored at ˜80° C. Then, the frozen brains werecoronally cut into 20-μm sections in a cryostat at −22° C. and every15^(th) slice was mounted on positively charged glass slides. Cerebralsections were stained with cresyl violet for 15 min. Each section wasphotographed. The areas of infarction were delineated on the basis ofthe relative lack of staining in the ischemic region and measured byusing NIH ImageJ software. Infarct area in each section was determinedas the total area of the contralateral hemisphere minus the non-affectedarea of the ipsilateral hemisphere.

Neurological evaluation. One hour after operation, as well as 24 h afterMCAO, animals were subjected to a series of motor tests performed. Theexaminations and scoring were as follows: 0, Normal; 1, Contralateralfront or rear limb flexion upon lifting of the whole animal by the tail;2, Circling to the contralateral side while walking and C-shaped lateralbending while hold by tail; 4 Circling to the contralateral side whilewalking and C-shaped lateral bending while hold by tail with limbflexion; 5, Comatose or moribund. The magnitude of the obtainedneuroscore is directly proportional to the severity of impairment.

EXAMPLE 21 NRP1-Trap Protects Against Cerebral Ischemia and Stroke

In order to assess the outcome of SEMA3A neutralization on cerebralischemia or stroke, adult (8-12 week-old) mice were subjected to thetransient middle cerebral artery occlusion (MCAO) model. Experimentaldetails are provided in Example 20. Briefly, following termination ofMCAO, mouse NRP1-trap (Trap-1, without FC,), 0.4 ug in 125 ul of PBS,(FIG. 19C, FIG. 20X-20Y; SEQ ID NO: 25) was administered to the tailvein (about 15 min after reperfusion had been started). In order tovisualize brain damage induced by MCAO, coronal cerebral sections werestained with cresyl violet. On each section, the unstained areacorresponded to the ischemic region of the brain (FIG. 18A). Measurementof these areas on serial coronal sections revealed that 24 h after MCAO,the infarcted zone constituted 48% of the ipsilateral hemisphere inoccluded mice compared to sham operated animals whose brains were notinjured. NRP1 treatment reduced brain damage; the infarct volume of theipsilateral hemisphere was decreased by 80% (FIGS. 18B,C).

Neurological impairment was assessed by neurological scoring of thepresence of limb flexion, C-shaped lateral bending of the body andcircling movements. MCAO mice that were not showing circling and bendingbehaviour 1 hour after operation were excluded from the further study(FIG. 18D). Forelimb or hindlimb flexion, C-shaped lateral bending ofthe body, circling movements were observed in mice subjected to MCAOcompared to sham operated animals. NRP1 treatment dramatically improvedneurological scores of ischemic mice by 60% compared to non-treated MCAOmice when evaluated 24 hours after surgery (FIG. 18E).

Taken together, these results show that inhibition of the NRP1 pathwayprotects against cerebral ischemia and stroke and reduce theneurological impairment associated with cerebral ischemia and stroke.

EXAMPLE 22 Neuropilin-Derived Traps Enhance Vascular Regeneration andPrevent Pathological Neovascularization in Ischemic Retinas in MouseModel of Diabetic Retinopathy and Retinopathy of Prematurity

Pathological vascular degeneration as well as pre-retinal vascularproliferation were studied using the well-established mouse model ofoxygen-induced proliferative retinopathy (OIR)(Smith et al., 1994). Thismodel is based on retinopathy of prematurity (ROP) and is regularly usedas a proxy for the proliferative (angiogenic phase) of diabeticretinopathy and ROP.

Nursing mothers and their pups were exposed to 75% oxygen from P7-P12.Both vaso-degenerative (assessed at P12) and vaso-proliferative(assessed at P17) phases are present and are highly reproducible makingevaluation of interventions on disease progression accurate and swift.Trap G (SEQ ID NO: 38), or Trap M (lacking the b2 and c domains, SEQ IDNO: 42), was injected into the vitreous at P12 (1 ul at 0.5 ug/ul).Dissected retinas were flatmounted and incubated overnight withfluoresceinated isolectin B4 (1:100) in 1 mM CaCl₂ to determine extentof avascular area or neovascularization area at P17. Avascular areaswere determined in lectin stained retinas as zones devoid of staining.Neovascularization was determined as areas of saturated lectin stainingwhich demarcates pre-retinal tufts (54, 55).

Trap G, was shown to effectively enhance vascular regeneration by over40% when compared to vehicle control (FIG. 23B). Similarly, Trap G wasshown to inhibit pathological neovascularization by ˜45% (FIG. 23C).Trap-M enhanced vascular regeneration by ˜60% (FIG. 23B) and inhibitedpathological neovascularization by ˜60% when compared to vehiclecontrols (FIG. 23C). Hence, Trap M, with compromised VEGF binding, moreeffectively prevents pathological angiogenesis and more readily leads toenhanced vascular regeneration in the ischemic retina.

EXAMPLE 23 Neuropilin-Derived Traps Decrease Vascular Leakage inDiabetic Retinas.

The influence of Traps on vascular leakage/permeability in diabeticretinopathy was also studied in the streptozotocin (STZ) model of Type 1diabetes. STZ (55 mg/kg) was administered over 5 consecutive days to ˜6week-old C57BL/6J mice and glycemia was monitored. Mice were considereddiabetic if their non-fasted glycemia was higher than 17 mM (300 mg/dL).Mice were administered intravitreally with 0.5 ug (0.5 ug/ul) of Trap G(SEQ ID NO: 38) or M (SEQ ID NO: 42) or with mouse anti-VEGF antibody(AF-493-NA, from R&D) at 6 and 7 weeks after STZ administration.Alternatively, mice were injected intravitreally at 12 and 13 weeks postSTZ and vascular permeability assessed at 14 weeks. Mice werehyperglycemic/diabetic at least 3 weeks prior to intravitreal injectionswith SEMA traps (see FIG. 24A) or anti VEGF antibody. Retinal vascularleakage was determined by Evans Blue assay at 8 weeks post STZinjections as follows. Retinal Evans Blue (EB) permeation was performedusing 3 retinas per reading. Evan Blue was injected at 45 mg/kgintravenously and allowed to circulate for 2 hours prior to retinalextraction. Evans Blue Permeation was quantified in retinas byfluorimetry (620 nm max absorbance-740 nm min absorbance (background)with a TECAN Infinite® M1000 PRO. Evan Blue Permeation (EBP) [measuredin uL/(grams*hour)] was calculated as follows: [EB (ug)/Wet retinalweight (g)]/[plasma EB (ug/uL)*Circulation time (hours)]. Evans Bluepermeation was expressed relative to controls.

Both Trap-G (SEQ ID NO: 38) and Trap-M (SEQ ID NO: 42) significantlyreduced vascular permeability by over 40% (FIG. 24B). The mouseanti-VEGF antibody (AF-493-NA) did not prevent vascular permeability atthis early stage. Trap G was effective at reducing vascular leakage aswas the anti-VEGF neutralizing Ab at P17 (FIG. 24C), *p<0.05, n=4, from12 animals.

EXAMPLE 24 Neuropilin-Derived Traps Decrease ChoroidalNeovascularization in Model of Age-Related Macular Degeneration

The effect of NRP1 trap G (SEQ ID NO: 38) on choroidalneovascularization (CNV) was determined in a mouse model of age-relatedmacular degeneration (AMD). To induce CNV and thus mimic wet AMD inmice, laser coagulations on 6-8 week old mice (1-2 disc diameters) wereperformed from the papillae using an Argon laser (532 nm) mounted on aCoherent slit lamp (400 mW, 50 ms and 50 μm) (Combadiere et al., 2007).Following laser burn, treated mice were injected intravitreally with 0.5ug of Trap G. Fourteen days (P14) later, choroids were radially incised,flat-mounted and stained with the endothelial cell markerfluoresceinated Isolectin B4 (animals were also optionally perfused withfluorescein dextran to visualize luminal vessels) and volumes of CNVwere measured by scanning laser confocal microscopy (Takeda et al.,2009).

Trap G was shown to significantly reduce choroidal neovascularization atday 14 post laser-burn (FIG. 25B).

The scope of the claims should not be limited by the preferredembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole.

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1. An ophthalmic pharmaceutical composition comprising (a) aneuropilin-1 (NRP1) polypeptide in an amount suitable to reduce eyeinflammation in a subject, the NRP1 polypeptide comprising an amino acidsequence having at least 90% identity with residues 22-424 of SEQ ID NO:107, wherein said NRP1 polypeptide does not comprise (i) the full b2domain or (ii) the full b2 and c domains, of native human NRP1; and (b)at least one carrier for ophthalmic administration.
 2. The compositionof claim 1, wherein said NRP1 polypeptide comprises an amino acidsequence having at least 95% identity with residues 22-424 of SEQ ID NO:107.
 3. The composition of claim 1, wherein said NRP1 polypeptidecomprises residues 22-424 of SEQ ID NO:
 107. 4. The composition of claim1, wherein said at least one carrier is an aqueous or oily carrier. 5.The composition of claim 1, wherein said at least one carrier is aphysiological saline buffer.
 6. The composition of claim 1, wherein saidcomposition is a suspension, solution or emulsion.
 7. The composition ofclaim 1, wherein said composition is an eye drop formulation.
 8. Thecomposition of claim 1, wherein said composition is an injectableformulation.
 9. The composition of claim 1, wherein said compositionfurther comprises a preservative.
 10. The composition of claim 8,wherein said composition is present in an injection device, an ampouleor a multi-dose container.
 11. A method for preparing the ophthalmicpharmaceutical composition of claim 1 comprising mixing: (a) the NRP1polypeptide comprising an amino acid sequence having at least 90%identity with residues 22-424 of SEQ ID NO: 107, wherein said NRP1polypeptide does not comprise (i) the full b2 domain or (ii) the full b2and c domains, of native human NRP1; with (b) the at least one carrierfor ophthalmic administration.
 12. A method for reducing ocularinflammation in a subject, comprising administering in the eye of thesubject an effective amount of the composition of claim 1.